Differential Effects of Botulinum Neurotoxin A on Bladder Contractile Responses to Activation of Efferent Nerves, Smooth Muscles and Afferent Nerves in Rats

Differential Effects of Botulinum Neurotoxin A on Bladder Contractile Responses to Activation of Efferent Nerves, Smooth Muscles and Afferent Nerves in Rats Ryosuke Takahashi, Takakazu Yunoki, Seiji Naito and Naoki Yoshimura* From the Departments of Urology, University of Pittsburgh School of Medicine (RT, NY), Pittsburgh, Pennsylvania, and Graduate School of Medical Sciences, Kyushu University (RT, TY, SN), Fukuoka, Japan

Purpose: To determine the mechanisms of botulinum neurotoxin A (Metabiologics, Madison, Wisconsin) induced inhibition of bladder activity we examined the effect of botulinum neurotoxin A on detrusor contractile responses to the activation of L-type voltage-gated Ca2⫹ channels, and efferent and afferent nerve terminals in the rat bladder. Materials and Methods: Rat bladder strips were incubated for 3 hours with different concentrations of botulinum neurotoxin A (0.3 to 100 nM). We examined the effect of botulinum neurotoxin A on detrusor contractility in response to activation of L-type voltage-gated Ca2⫹ channels, and efferent and afferent nerve terminals induced by 70 mM KCl, electrical field stimulation and 1 ␮M capsaicin, respectively. Results: Botulinum neurotoxin A inhibited electrical field stimulation induced contractions at a concentration of 10 nM or higher. The maximal inhibition at 100 nM was 70% compared to that of control strips. KCl induced contractions, which were sensitive to nifedipine, were significantly inhibited by incubation with botulinum neurotoxin A at a concentration of 3 nM or higher. Maximal inhibition at 100 nM was 30% compared to that of control strips. Capsaicin induced contractions were not inhibited by 3-hour incubation but they were significantly inhibited by overnight incubation with 100 nM botulinum neurotoxin A (30% compared to control strips). Carbachol induced contractions were not altered by incubation with botulinum neurotoxin A. Conclusions: The order of inhibitory potency of botulinum neurotoxin A was efferent nerve terminals ⬎L-type voltage-gated Ca2⫹ channels ⬎afferent nerve terminals. Since the inhibitory effects on L-type voltage-gated Ca2⫹ channels and efferent nerve terminals were observed at similar botulinum neurotoxin A concentrations, the inhibitory effect of botulinum neurotoxin A on L-type voltagegated Ca2⫹ channels may have an important role in regulating and stabilizing bladder activity.

Abbreviations and Acronyms

␣,␤-MetATP ⫽ ␣,␤-methylene ATP ATP ⫽ adenosine triphosphate BoNT/A ⫽ botulinum neurotoxin A CCh ⫽ carbachol EFS ⫽ electrical field stimulation OAB ⫽ overactive bladder SNAP-25 ⫽ synaptosomalassociated protein with molecular weight 25 kDa TTX ⫽ tetrodotoxin VGCC ⫽ voltage-gated Ca2⫹ channel Submitted for publication February 16, 2012. Study received institutional animal care and use committee approval. Supported by National Institutes of Health Grants (DK057267 and DK088836), Department of Defense Grant SC100134 and Paralyzed Veterans of America Grant 2793. * Correspondence: Department of Urology, University of Pittsburgh School of Medicine, Suite 700 Kaufmann Medical Building, 3471 Fifth Ave., Pittsburgh, Pennsylvania 15213 (telephone: 412-6924137; FAX: 412-692-4380; e-mail: [email protected]).

Key Words: urinary bladder; muscle contraction; botulinum toxins, type A; calcium channels; efferent pathways BOTULINUM neurotoxin A has been used to treat neurogenic as well as idiopathic OAB that fails to respond to conservative management and antimuscarinic drugs. The primary in-

hibitory mechanism of BoNT/A is considered to be blockage of acetylcholine exocytosis from efferent nerve terminals, leading to the suppression of neurogenic contractile and/or myogenic

0022-5347/12/1885-1993/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION

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behavior in patients with OAB.1 BoNT/A cleaves specific sites of SNAP-25 in cytoplasm, which prevents the assembly of synaptic fusion complex proteins and blocks exocytosis.1 However, the efficacy of BoNT/A treatment sometimes exceeds that expected for simple detrusor muscle paralysis.2 Some patients report major sensory benefits, including decreased urgency and pain. Also, recently there has been increasing evidence of the effect of BoNT/A on bladder afferent pathways. In cultured rat dorsal root ganglion neurons BoNT/A induced delayed, long lasting inhibition of substance P release after the initial cleavage of SNAP-25.3 In isolated rat bladder BoNT/A application inhibited the stimulated release of calcitonin-gene related peptide from afferent nerve terminals.4 To our knowledge the direct effect of BoNT/A on bladder smooth muscle has yet to be reported. In pancreatic ␤ cells Ji et al noted that the COOHterminal (amino acid 198 –206) domains of SNAP-25 (SNAP-25198 –206) inhibited L-type VGCCs.5 In our preliminary study BoNT/A cleaved SNAP-25, while SNAP-25198 –206 (BoNT/A cleaved product) inhibited L-type VGCCs in rat bladder smooth muscle.6 However, to our knowledge the functional inhibitory effect of BoNT/A on L-type VGCCs in bladder smooth muscle has not yet been examined. In the current study we first investigated the functional effect of BoNT/A on detrusor contractile responses to L-type VGCC activation. We then examined the inhibitory effect of BoNT/A on detrusor contractile responses to the activation of efferent and afferent nerve terminals, and evaluated the relative importance of the inhibitory effects of BoNT/A on these 3 mechanisms (L-type VGCCs, and efferent and afferent nerve terminals).

MATERIALS AND METHODS Tissue Preparation Normal female Sprague-Dawley® rats weighing 200 to 250 gm were used. All animal experiments were done in accordance with institutional guidelines and approved by the University of Pittsburgh animal care and use committee. The bladder was removed with the rat under isoflurane (2%) anesthesia and placed in modified Krebs solution composed of 143 mM Na⫹, 5.9 mM K⫹, 2.5 mM Ca2⫹, 1.2 mM Mg2⫹, 127.7 mM Cl–, 1.2 mM SO42⫺, 1.2 mM PO43⫺, 25 mM HCO3⫺ and 11 mM glucose, bubbled with 95% O2 and 5% CO2 to attain pH 7.4. Tissue from the bladder body above the urethral orifices was longitudinally cut under a binocular microscope into strips approximately 0.5 ⫻ 0.5 ⫻ 7 mm and weighing 1 to 2 mg. The mucosa containing the urothelium was removed with microscissors under a binocular microscope to focus on the direct effects of BoNT/A on detrusor muscle.

Bladder Strip Tension Measurement Bladder strips were incubated for 3 hours at 37C in modified Krebs solution with or without different concentrations of full-length BoNT/A (0.3 to 100 nM). Tension development was measured in an organ bath. Each strip was attached to isometric force displacement transducers using a Myobath carrier amplifier (World Precision Instruments, Sarasota, Florida) and mounted vertically in a 25 ml bath containing modified Krebs solution. The initial load was set to 1.0 gm. Strips were equilibrated in modified Krebs solution for at least 1 hour before measurement. All data were compared with those on vehicle incubated control muscle strips. EFS was performed in preparations mounted between 2 platinum electrodes in an organ bath with a Grass S88 stimulator (Grass Technologies, West Warwick, Rhode Island). The intrinsic nerves were stimulated with rectangular 20 V pulses 5 milliseconds in duration at a frequency of 0.5 to 64 Hz. Trains of pulses lasted 2 seconds and the stimulation interval was 120 seconds.

Drugs and Solutions High K solutions were made by equimolar substitution of KCl for NaCl. All solutions were gassed with a mixture of 95% O2 and 5% CO2 (pH 7.4 at 37C). CCh, TTX, capsaicin, nifedipine, atropine and ␣,␤-MetATP were obtained from Sigma®.

Statistical Analysis Peak values of detrusor contractions were measured and averaged in each group of animals for statistical analysis. All data are shown as the mean ⫾ SEM. The unpaired Student t test and 1-way ANOVA were used to determine statistical differences between 2 groups and among more than 2 groups, respectively, with p ⬍0.05 considered significant. A 4 parameter logistic model was used to fit the sigmoidal curve to the concentration response of each drug.7 Data were collected and analyzed using PowerLab®.

RESULTS BoNT/A Effect on Bladder Contractile Responses Activation of L-type VGCCs of rat bladder smooth muscle. To examine the functional effect of BoNT/A on L-type VGCCs, changes in the contraction elicited by 70 mM KCl were evaluated. KCl induces smooth muscle membrane depolarization and is supposed to activate nifedipine sensitive VGCCs. The developed tension induced by 70 mM KCl was evaluated at a peak contraction. All experiments were performed in the presence of 1 ␮M TTX to exclude the effect of nerve activation. Figure 1, A and B shows representative recordings of 70 mM KCl induced contractions without and with, respectively, 30 nM BoNT/A incubation. Contractile responses induced by 70 mM KCl were dose dependently inhibited by incubation with BoNT/A (fig. 1, C). The significant inhibitory effect of BoNT/A on 70 mM KCl induced contractions was observed at a concentration of 3 nM or higher. Maximal inhibi-

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KCl was applied in the presence of nifedipine. The peak contraction induced by the first 70 mM KCl was considered 100% and the second 70 mM KCl induced peak contraction was evaluated. The application of 300 nM nifedipine inhibited 70 mM KCl induced contraction by a mean of 70.6% ⫾ 3.08% in 7 preparations (fig. 1, D), indicating that most contraction induced by 70 mM KCl was mediated through nifedipine sensitive L-type VGCCs. In addition, the application of 10 and 30 nM nifedipine inhibited the 70 mM KCl induced contraction by 22.0% ⫾ 1.95% and 47.8% ⫾ 2.07%, respectively, in 5 preparations each. These results suggest that the inhibitory potency of BoNT/A is equal to that of nifedipine at concentrations between 10 and 30 nM.

Figure 1. Mean ⫾ SEM effects of different concentrations of BoNT/A or nifedipine on membrane depolarization induced contraction of bladder muscle strips elicited by KCl application. Representative recordings of 70 mM KCl induced contractions without (A) or with (B) 30 nM BoNT/A incubation. Effect of different concentrations of BoNT/A (0.3 to 100 nM) on 70 mM KCl induced contraction in 10 preparations (C). NS, not significant. Single asterisk indicates p ⬍0.05 vs time matched control. Double asterisks indicate p ⬍0.01 vs time matched control. Effect of different concentrations of nifedipine (3 to 300 nM) on 70 mM KCl induced contraction in 5 to 7 preparations (D).

tion at 100 nM was about 30% compared to time matched control strips in 10 preparations each (mean 1.56 ⫾ 0.11 vs 0.99 ⫾ 0.06 gm, p ⬍0.01, fig. 1, C). In the control group we also examined the effect of nifedipine, a specific inhibitor of L-type VGCCs, on the contractile response induced by 70 mM KCl. After the first application of 70 mM KCl followed by a single washout, nifedipine at different concentrations (3, 10, 30, 100 or 300 nM) was added to the bath. After 20-minute incubation, the second 70 mM

Activation of efferent nerve terminals. To examine the effect of BoNT/A on neurotransmitter release from efferent nerve terminals, changes in EFS induced contractions were evaluated. Of EFS induced contractions 80% were inhibited by treatment with 1 ␮M TTX (fig. 2, A). This indicates that the greater part of EFS induced contractions is caused by nerve stimulation with the opening of TTX sensitive voltage-gated Na⫹ channels. The contractile responses induced by EFS were dose dependently inhibited by 3-hour incubation with BoNT/A (fig. 3). The significant inhibitory effect of BoNT/A on EFS induced contractions was observed at a concentration of 10 nM or higher. The maximal inhibition at 100 nM was about 70% compared to time matched control strips (mean 0.87 ⫾ 0.17 gm in 10 preparations vs 0.22 ⫾ 0.06 gm in 6, p ⬍0.05, fig. 3, F). The inhibitory effect of 30 nM BoNT/A on EFS induced contractions was similar to that of 1 ␮M TTX (fig. 2, A). We next examined the contributions of cholinergic and purinergic receptor activation in EFS induced contractions. After the first application of several frequencies (0.5 to 64 Hz) of EFS at 120-second intervals, atropine (muscarinic receptor antagonist) or atropine plus ␣,␤-MetATP (P2X purinergic receptor antagonist) was added to the bath. After a 20minute incubation, the second EFS was applied in the presence of these inhibitors. The peak contraction induced by the first 64 Hz EFS was considered 100% and the second EFS induced peak contractions were evaluated. Atropine was used at a concentration of 1 ␮M, which was confirmed to completely block 30 ␮M CCh induced contraction (data not shown). This indicated that this concentration of atropine would almost completely block cholinergic receptor activation. The application of 1 ␮M atropine for 20 minutes significantly inhibited EFS induced contractions (fig. 2, B). The inhibitory effect of atropine was more prominent for high than for low

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Figure 2. Mean ⫾ SEM effect of TTX, atropine or ␣,␤-MetATP on EFS induced contraction in 6 control strips. Effect of 30 nM BoNT/A or 1 ␮M TTX on EFS induced contraction vs time matched control strips (A). Effect of 1 ␮M atropine, 1 ␮M atropine plus ␣,␤-MetATP or 1 ␮M TTX on EFS induced contraction vs time matched controls (B). NS, not significant. Single asterisk indicates p ⬍0.05 vs atropine treated strips. Double asterisks indicate p ⬍0.01 vs time matched control strips. Single pound sign indicates p ⬍0.05. Double pound signs indicate p ⬍0.01.

frequency stimulation (39% inhibition of control contraction at 2 Hz and 55% inhibition at 64 Hz). The residual atropine resistant contraction was evaluated by desensitization of P2X purinergic receptors with ␣,␤-MetATP, which was applied at a

Figure 3. A to F, mean ⫾ SEM effect of different BoNT/A concentrations (0.3 to 100 nM) on EFS induced contraction in 6 preparations. NS, not significant. Asterisk indicates p ⬍0.05 vs time matched control.

concentration of 10 ␮M 2 or 3 times at 10-minute intervals in the presence of atropine until the contraction caused by this agent was absent. The application of atropine plus ␣,␤-MetATP caused a further decrease in EFS induced contraction, which was the same level as that of 1 ␮M TTX treatment. This suggests that BoNT/A suppresses the release of acetylcholine and ATP from efferent nerve terminals to decrease EFS induced contractile responses. Activation of afferent nerve terminals. We previously reported that capsaicin induces bladder smooth muscle contraction by stimulating the TRPV1 receptor expressed on afferent nerve terminals, followed by the release of neuropeptides that can activate tachykinin NK1 and/or NK2 receptors on bladder smooth muscle.8 Therefore, to examine the effect of BoNT/A on afferent nerve terminals we evaluated changes in capsaicin (1 ␮M) induced contractions. The application of 1 ␮M capsaicin produced a phasic contraction (fig. 4, A and B). In the 3-hour incubation protocol capsaicin induced contractions were not altered even at the highest concentration of 100 nM BoNT/A (fig. 4, A to D). There was no significant difference between the 2 groups in 10 preparations each (mean 0.59% ⫾ 0.06% vs 0.57% ⫾ 0.06% and 42.7% ⫾ 2.0% vs 42.6% ⫾ 3.2%, respectively, fig. 4, C and D). Welch et al previously reported that rat ganglion substance P secretion was not significantly inhibited until 4 hours of incubation with 100 nM BoNT/A and a greater degree of inhibition was observed after 8 hours of incubation.3 Therefore, we next tried overnight incubation with 100 nM BoNT/A. Under this condition capsaicin induced contractions were significantly inhibited by approximately 30% compared to time matched control strips (mean 0.35 ⫾ 0.03 gm in 9 preparations vs 0.23 ⫾ 0.03 gm in 10, p ⬍0.05, fig. 4, E to G). Even when values were corrected with

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between these 2 groups in 10 preparations each (mean 2.09 ⫾ 0.19 vs 1.78 ⫾ 0.11 gm, p ⫽ 0.19, and 1.79% ⫾ 5.12% vs 1.60% ⫾ 0.10%, p ⫽ 0.25, fig. 5, B and C). In the control group we also examined the effect of nifedipine on the contractile response induced by CCh. After the first cumulative application of CCh (10 nM to 30 ␮M), followed by washout twice, nifedipine at different concentrations (10, 30, 100 or 300 nM) was added to the bath. After 20-minute incubation, the second CCh was cumulatively applied in the presence of nifedipine. The peak contraction induced by the first 30 ␮M CCh was considered 100% and the second cumulative CCh induced contractions were evaluated. Contractile responses induced by CCh were dose dependently inhibited by nifedipine treatment (fig. 5, D).

DISCUSSION There are 3 major findings in our study. 1) BoNT/A has a functional inhibitory effect on neurotransmitter release from efferent and afferent nerve terminals as well as on L-type VGCCs expressed on bladder smooth muscle. 2) The order of the inhibitory potency of BoNT/A on bladder contractile responses to the activation of these 3 pathways is efferent nerve terminals ⬎L-type VGCC ⬎afferent nerve terminals. 3) The inhibitory effect of BoNT/A on L-type VGCCs and efferent nerve terminals occur at similar BoNT/A concentrations. To our knowledge this is

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Figure 4. Mean ⫾ SEM effect of 100 nM BoNT/A on capsaicin induced contraction of 9 or 10 preparations. Strips were pretreated with 100 nM BoNT/A for 3 hours (A to D) or overnight (E to H). Representative recordings of 1 ␮M capsaicin induced contraction without (A and E) or with (B and F) incubation with 100 nM BoNT/A for 3 hours (A and B) or overnight (E and F). Results were evaluated at peak contraction induced by 1 ␮M capsaicin (C and G). Peak contractions induced by 1 ␮M capsaicin are shown as percent of peak contraction induced by 1 ␮M CCh (D and H). g, gm. NS, not significant. Asterisk indicates p ⬍0.05 vs time matched control.

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the first report to clarify the functional inhibitory effect of BoNT/A on bladder smooth muscle and compare the inhibitory effect of BoNT/A on 3 pathways (efferent and afferent nerve terminals, and bladder smooth muscle) under the controlled condition. A previous study showed different results of negative effects of BoNT/A incubation for 72 hours on EFS induced detrusor contraction in rodents.9 The discrepancy between those results and ours may be due to differences in species (rats vs mice/guinea pigs), the source of botulinum toxins and the larger muscle strips used in that study. BoNT/A had a smaller inhibitory effect on 70 mM KCl induced contractions than on EFS induced contractions (30% vs 70%) (figs. 1 and 3). However, the inhibitory effects of BoNT/A on these 2 contractile responses were observed at similar BoNT/A concentrations (3 and 10 nM, respectively), suggesting that the inhibitory effect of BoNT/A on L-type VGCC mediated direct detrusor contraction may function in the clinical setting, where BoNT/A is expected to suppress transmitter release from efferent nerve terminals. Previous studies described the close relationship between OAB symptoms and L-type VGCC function. For example, spontaneous contractions are blocked by dihydropyridine L-type Ca channel blockers, such as nifedipine, in guinea pigs10 and rats with bladder overactivity.11 Also, myogenic spontaneous contraction causes an involuntary increase in intravesical pressure, which results in urinary incontinence.12 Recently, Leiria et al reported that extracellular Ca2⫹ influx through L-type VGCCs has a major role in the overactive detrusor in diabetic mice.13 These findings raise the possibility that the inhibitory effect of BoNT/A on L-type VGCCs may have an important role in regulating and stabilizing detrusor smooth muscle contractility in treatment for OAB. In the current study the mechanism underlying the inhibitory effects of BoNT/A on L-type VGCCs was not explored. However, Ji et al reported that SNAP-25198 –206 (BoNT/A cleaved product) decreased L-type Ca2⫹ currents in pancreatic islet ␤-cells.5 In the bladder BoNT/A cleaves specific sites on SNAP-25.1 These data support the possibility that the same mechanism might function in bladder smooth muscle. To evaluate the inhibitory effect of BoNT/A on afferent nerve terminals, we examined the capsaicin induced contraction of detrusor muscle because capsaicin induced detrusor contraction is predominantly induced by the activation of neurokinin receptors in response to neuropeptides released from afferent nerves.8,14 However, BoNT/A action on other compartments, such as smooth muscle and blood vessels, cannot be completely excluded. Results demonstrated that longer incubation (over-

night) with BoNT/A was needed to inhibit capsaicin induced contractions (fig. 4), indicating that a high tissue concentration of BoNT/A may be necessary to suppress neurotransmitter release from afferent nerve terminals. Therefore, we conclude that suppressing neurotransmitter release from afferent nerve terminals seems to contribute to a lesser degree to BoNT/A mediated inhibition of bladder activity compared with the other 2 inhibitory mechanisms (efferent nerve terminals and L-type VGCCs of bladder smooth muscle). Our results must be interpreted with caution because our experiments were designed to investigate BoNT/A effects on the contraction of detrusor muscle strips without the mucosa containing urothelium and suburothelial interstitial cells, which are each known to affect bladder afferent activity.15 For example, Khera et al reported that BoNT/A inhibits ATP release from urothelium.16 BoNT/A also has an inhibitory effect on ATP release from efferent nerve terminals (fig. 2, B). These 2 mechanisms for decreasing ATP release would have an inhibitory effect on ATP induced indirect activation of afferent nerves. In addition, Apostolidis et al reported that BoNT/A decreased expression levels not only of TRPV1 but also of P2X3 purinergic receptors in suburothelial nerve fibers in patients with OAB.17 In the clinical field BoNT/A often attenuates urgency and pain before urodynamic improvement. Neurologists have long recognized decreased pain in patients after intramuscular injection of BoNT/A and this benefit has often been reported before there is evidence of muscle relaxation.18 Taken together, it is possible that the overall (direct plus indirect) inhibitory effects of BoNT/A on afferent nerve activity would be more significant than we noted. Nevertheless, this study reveals that the inhibitory effects of BoNT/A on afferent nerves are less than those on efferent nerves and detrusor muscle. However, the lower density of afferent nerve endings in the detrusor compared to the suburothelium and/or limited tissue penetration of BoNT/A due to its heavy molecular weight might have affected the results. Neuromuscular transmission in the healthy human bladder is greater than 95% atropine sensitive but the purinergic component can increase to up to 40% in cases of interstitial cystitis19 or idiopathic detrusor instability, possibly because of altered P2X receptor expression.20 Lawrence et al reported that cholinergic and purinergic signals are equally susceptible to BoNT/A.21 Our results are consistent with this report (fig. 2, B). Therefore, these results suggest that the effectiveness of BoNT/A for treating patients with OAB who are nonresponders to antimuscarinics may be related to the inhibitory effect on ATP release from efferent nerve terminals.

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L-type VGCC activation is reportedly involved in CCh induced detrusor contraction. Thus, we initially speculated that BoNT/A, which inhibited L-type VGCCs, as evidenced by the significant reduction of 70 mM KCl induced contraction (fig. 1, C), could decrease CCh induced detrusor muscle contraction. However, CCh induced contraction was not significantly inhibited by BoNT/A treatment even at the highest concentration of 100 nM (fig. 5, C), although CCh induced contraction was dose dependently inhibited by nifedipine (fig. 5, D). Kishii et al reported that the inhibitory effects of Ca2⫹ channel antagonists or removal of extracellular Ca2⫹ on carbachol induced contraction is less than that on high K⫹ induced contractions.22 Wu et al also reported that activation of muscarinic cholinergic receptors is not associated with membrane potential depolarization or the opening of L-type VGCCs but it can modulate L-type VGCCs via secondary voltage dependent membrane events, such as the suppression of K⫹ channel activity.23 There-

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fore, these results suggest that the contribution of L-type VGCCs to CCh induced contraction is not as notable as that of KCl induced contraction. Another possibility to explain this discrepancy is that Ca2⫹ release from the sarcoplasmic reticulum may make up for the decreased Ca2⫹ influx through L-type VGCCs. The increase in the intracellular Ca2⫹ concentration occurs through Ca2⫹ influx and also by Ca2⫹ release from intracellular Ca2⫹ stores.24 Further studies are needed to clarify these points.

CONCLUSIONS The current study shows that the order of inhibitory potency of BoNT/A is efferent nerve terminals ⬎Ltype VGCCs on smooth muscles ⬎afferent nerve terminals. The study also shows that BoNT/A can inhibit acetylcholine and ATP release from efferent nerve terminals. Thus, BoNT/A has multiple inhibitory mechanisms affecting bladder function, which would enhance the efficacy of BoNT/A treatment for overactive bladder.

REFERENCES 1. Chancellor M, Fowler CJ, Apostolidis A et al: Drug insight: biological effects of botulinum toxin A in the lower urinary tract. Nat Clin Pract Urol 2008; 5: 319. 2. Apostolidis A, Dasgupta P and Fowler CJ: Proposed mechanism for the efficacy of injected botulinum toxin in the treatment of human detrusor overactivity. Eur Urol 2006; 49: 644. 3. Welch MJ, Purkiss JR and Foster KA: Sensitivity of embryonic rat dorsal root ganglia neurons to clostridium botulinum neurotoxins. Toxicon 2000; 38: 245. 4. Rapp DE, Turk KW, Bales GT et al: Botulinum toxin type A inhibits calcitonin gene-related peptide release from isolated rat bladder. J Urol 2006; 175: 1138. 5. Ji J, Yang SN, Huang X et al: Modulation of L-type Ca2⫹ channels by distinct domains within SNAP-25. Diabetes 2002; 51: 1425.

anandamide in muscle strips isolated from the rat urinary bladder. Eur J Pharmacol 2007; 570: 182.

lium after chronic spinal cord injury. Neurochem Int 2004; 45: 987.

9. Howles S, Curry J, McKay I et al: Lack of effectiveness of botulinum neurotoxin A on isolated detrusor strips and whole bladders from mice and guinea-pigs in vitro. BJU Int 2009; 104: 1524.

17. Apostolidis A, Popat R, Yiangou Y et al: Decreased sensory receptors P2X3 and TRPV1 in suburothelial nerve fibers following intradetrusor injections of botulinum toxin for human detrusor overactivity. J Urol 2005; 174: 977.

10. Mostwin JL: The action potential of guinea pig bladder smooth muscle. J Urol 1986; 135: 1299. 11. McCarthy CJ, Zabbarova IV, Brumovsky PR et al: Spontaneous contractions evoke afferent nerve firing in mouse bladders with detrusor overactivity. J Urol 2009; 181: 1459. 12. Brading AF and Turner WH: The unstable bladder: towards a common mechanism. Br J Urol 1994; 73: 3. 13. Leiria L, Monica FZT, Carvalho FDGF et al: Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2⫹ channels. Br J Pharmacol 2011; 163: 1276.

6. Yunoki T, Naito S and Yoshimura N: The effects of botulinum neurotoxin-A on L-type and T-type voltage-gated Ca2⫹ current in detrusor smooth muscles. Presented at annual meeting of International Continence Society/International Urogynecological Association, Toronto, Ontario, Canada, August 22–27, 2010, program 599.

14. Maggi CA, Patacchini R, Santicioli P et al: Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission. Br J Pharmacol 1991; 103: 1535.

7. De Lean A, Munson PJ and Rodbard D: Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 1978; 235: E97.

15. Yoshimura N and Chancellor MB: Physiology and pharmacology of the bladder and urethra. In: Campbell-Walsh Urology, 10th ed. Edited by AJ Wein, LR Kavoussi, AC Novick et al. Philadelphia: Elsevier Saunders 2012; vol 3, sect XIV, chap 60, p 1786.

8. Saitoh C, Kitada C, Uchida W et al: The differential contractile responses to capsaicin and

16. Khera M, Somogyi GT, Kiss S et al: Botulinum toxin A inhibits ATP release from bladder urothe-

18. Relja M and Telarovic S: Botulinum toxin type-A and pain responsiveness in cervical dystonia: a dose response study. Mov Disord 2005; 20: P77. 19. Palea S, Artibani W, Ostardo E et al: Evidence for purinergic neurotransmission in human urinary bladder affected by interstitial cystitis. J Urol 1993; 150: 2007. 20. O’Reilly BA, Kosaka AH, Knight GF et al: P2X receptors and their role in female idiopathic detrusor instability. J Urol 2002; 167: 157. 21. Lawrence GW, Aoki KR and Dolly JO: Excitatory cholinergic and purinergic signaling in bladder are equally susceptible to botulinum neurotoxin A consistent with co-release of transmitters from efferent fibers. J Pharmacol Exp Ther 2010; 334: 1080. 22. Kishii K, Hisayama T and Takayanagi I: Comparison of contractile mechanisms by carbachol and ATP in detrusor strips of rabbit urinary bladder. Japan J Pharmacol 1992; 58: 219. 23. Wu C, Sui G and Fry CH: The role of the L-type Ca2⫹ channel in refilling functional intracellular Ca2⫹ stores in guinea-pig detrusor smooth muscle. J Physiol 2002; 583.2: 357. 24. Andersson KE and Arner A: Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 2003; 84: 935.