- Email: [email protected]
Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: Effect of botulinum toxin A
Neurochemistry International 47 (2005) 291–297 www.elsevier.com/locate/neuint
Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: Effect of botulinum toxin A Christopher P. Smith, Vijaya M. Vemulakonda, Susanna Kiss, Timothy B. Boone, George T. Somogyi * Scott Department of Urology, Baylor College of Medicine, One Baylor Plaza, Alkek N720, Houston, TX 77030, USA Received 7 January 2005; received in revised form 10 April 2005; accepted 11 April 2005 Available online 20 June 2005
Abstract The effects of mechanoreceptor stimulation and subsequent ATP release in cyclophosphamide evoked chronic bladder inflammation was examined to demonstrate: (1) whether inflammation modulates ATP release from bladder urothelium and (2) whether intravesical botulinum toxin A administration inhibits urothelial ATP release, a measure of sensory nerve activation. ATP release was measured from rat bladders in a Ussing chamber, an apparatus that allows one to separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Cystometry was utilized to correlate changes in ATP release with alterations in the frequency of voiding and non-voiding bladder contractions, in vivo measures of bladder afferent activity. The resting urothelial release of ATP was not significantly affected by either cyclophosphamide or botulinum toxin A treatment. However, evoked ATP release following hypoosmotic stimulation was significantly increased (i.e. 94%) in chronic cyclophosphamide treated bladder urothelium compared to control bladders. In addition, botulinum toxin A treatment significantly reduced hypoosmotic shock induced ATP release in cyclophosphamide treated animals by 69%. Cystometry revealed that cyclophosphamide and botulinum toxin A treatments altered non-voiding (i.e. cyclophosphamide increased, botulinum toxin A decreased) but not voiding contraction frequency suggesting that alterations in urothelial ATP release selectively diminished underlying bladder C-fiber nerve activity. Finally, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side implying that its effects were confined to the urothelial side of the bladder preparation. # 2005 Elsevier Ltd. All rights reserved. Keywords: Botulinum toxin; Bladder; Inflammation; Interstitial cystitis; Ussing chamber; ATP; Urothelium
1. Introduction Chronic inflammatory conditions have been shown to lower the pain threshold and increase perceptions of pain. Inflammation in visceral organs causes increased sensitivity to noxious and non-noxious stimulation (Habler et al., 1993). Cyclophosphamide (CYP) induced cystitis, an animal model of chronic bladder inflammation, has been shown to increase voiding frequency and induce bladder hyperactivity both acutely and chronically (Maggi et al., 1992; Vizzard, 2000).
* Corresponding author. Tel.: +1 713 798 3541; fax: +1 713 798 6454. E-mail address: [email protected] (G.T. Somogyi). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.04.021
ATP is known to be a prominent neurotransmitter at both efferent (Vizi et al., 1997) and sensory sites within the peripheral nervous system. Recent findings support the notion that epithelial tissues including urothelium may be involved in bladder sensory mechanisms (Ferguson et al., 1997; Ferguson, 1999; Birder et al., 2002). For example, activation of the urothelium by pressure, stretch or hypoosmotic shock releases ATP from the urothelial cells, both in cell culture and an in vitro system where an Ussing chamber is used to separate the urothelial and serosal sides (Ferguson et al., 1997; Khera et al., 2004). In this context, the increase in ATP release can be used as a marker for activation of the bladder urothelium. Prior investigations have demonstrated that ATP release from cultured urothelial cells is increased in patients suffering from interstitial
292
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
cystitis (IC) (Sun et al., 2001), a bladder disorder characterized by chronic inflammation and pain and thought to result, in part, from alterations in urothelial and afferent nerve function (Steers and Tuttle, 1997). Botulinum toxin A (BTX-A) inhibits neurotransmitter release from nerve terminals by inhibiting the fusion of the intracytoplasmic vesicle with the plasma membrane (Schiavo et al., 1993). We have previously demonstrated that BTX-A inhibits acetylcholine and norepinephrine release from efferent nerve terminals in the lower urinary tract (Smith et al., 2003). However, recent studies suggest that BTX may also exert a considerable inhibitory action on the sensory nervous system. For example, BTX-A has been shown to decrease neuropeptide release from rat dorsal root ganglia (Welch et al., 2000) and inhibit ATP release from the epithelium of intestine as well as the urothelium of neurogenic bladders (Khera et al., 2004). Furthermore, in vivo models of somatic (Cui et al., 2004) and visceral inflammation (Vemulakonda et al., 2005) and irritation (Chuang et al., 2004) support an anti-nociceptive effect of BTX-A. In this study, we evaluated the effect of chronic inflammation on hypoosmotic shock-induced ATP release from bladder urothelium using the previously described modified Ussing chamber model (Khera et al., 2004). Furthermore, we analyzed afferent effects of intravesically instilled BTX-A using ATP release as a marker under these noxious circumstances. Experiments were also conducted utilizing cystometry to correlate the in vitro changes in ATP release with the in vivo effects of chronic bladder inflammation and intravesical BTX-A instillation. A preliminary report of these results has been published (Vemulakonda et al., 2004).
2. Materials and methods 2.1. Preparation Female Sprague–Dawley rats weighing 200–250 g were divided by intravesical instillation and intraperitoneal injection into four groups (n = 5–6 per group) for both in vivo and in vitro experiments. (1) Saline instilled and saline injected (Control). (2). BTX-A instilled and saline injected (BTX). (3) Saline instilled and CYP injected (CYP). (4) BTX-A instilled and CYP injected (CYP + BTX). The effectiveness of chronic CYP treatment as well as the inhibitory effect of BTX-A instillation was evaluated with bladder cystometry by monitoring the frequency of voiding and non-voiding bladder contractions. All animal experiments were carried out in accordance with the National Institutes of Health guideline. 2.2. Experimental protocol On day 0, under isoflurane anesthesia rat bladders were filled with 1 ml of 1% protamine sulfate (Sigma, St. Louis,
MO) transurethrally for 30 min to disrupt the integrity of the urothelial barrier (Niku et al., 1994). The protamine sulfate was drained and the bladder was instilled either with 1 ml of 20 units BTX-A (Botox1, Allergan, Irvine, CA) or saline for 30 min (Khera et al., 2004; Vemulakonda et al., 2005). One unit of Botox1 equals the calculated intraperitoneal LD50 in mice (Botox1 package insert). Chronic cystitis was induced by injecting 150 mg/kg CYP (Sigma) intraperitoneally every third day for a total of three injections while control animals received the same volume of i.p. saline injection. Both the in vivo and in vitro experiments were performed on day 10. 2.2.1. Measurement of in vivo bladder contractions On the tenth day, a suprapubic catheter was placed through the bladder dome under isoflurane anesthesia. After recovery from anesthesia, the animals were restrained (Universal Rodent Restrainer, Harvard Apparatus, Holliston, MA) and the bladder was perfused with saline at a rate of 0.04 ml/min using a syringe pump (KDS, New Hope, PA). Bladder contractions and voided urine were measured with a pressure and force transducer system (WPI, Sarasota, FL), respectively, as described in earlier studies (Vemulakonda et al., 2005). The frequency of non-voiding or voiding bladder contractions (i.e. contractions where urine was expelled) was calculated and expressed as contractions/h. An increase in contraction frequency represented an increase in afferent nerve activity and vice-versa. The frequency of both types of bladder contractions was calculated using the Windaq playback program (WindaqEx, DATAQ, Akron, OH). 2.2.2. Measuring urothelial ATP release After completion of cystometrogram evaluation, animals were sacrificed and bladders were removed and mounted in an Ussing chamber, a tissue bath that consists of two parts separated by the bladder tissue cut opened where one chamber faces the urothelial side and the other chamber faces the serosal side of the bladder (for details see Khera et al., 2004). The serosal and urothelial hemichambers were separately perfused with oxygenated preheated 37 8C Krebs solution (mm/l; NaCl 113, KCl 4.7, CaCl2 1.25, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11.5) at a rate of 0.5 ml/ min with a peristaltic pump (Rainin, Minipuls2, Woburn, MA) for 1 h and 5 min effluents were collected using a fraction collector (Gilson, Middleton, WI). After taking two baseline samples, the urothelial surface was exposed to hypoosmotic Krebs solution (i.e. NaCl concentration was reduced by 40% from 113 to 67.8 mM) for 6 min and then perfused with isoosmotic Krebs solution for another 25 min. A 50 ml aliquot from each 5 min effluent was placed into a luminometer (Turner, Sunnyvale, CA) and the ATP content was measured by using the luciferin-luciferase assay. The luminescence of the collected samples was compared to that of standard ATP and the basal and evoked release was calculated. The average of the first and last samples in the
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
293
course of the perfusion was considered, in most cases, as the basal release of urothelial ATP and was standardized to the bladder surface area (i.e. fmol/cm2). Evoked release of ATP by hypoosmotic stimulation was expressed as area computed from the percent increase over the basal release of ATP. 2.3. Drugs Cyclophosphamide, protamine sulfate, the ATP bioluminescent assay kit and all constituents of Krebs solution were purchased from Sigma. BTX-A (Botox1) was a generous gift from Allergan Inc. (Irvin, CA). 2.4. Statistical analysis Statistical analysis was performed using Prism3 software (Graph Pad, San Diego, CA). The mean and standard error of the mean were calculated for each series of measurements. The statistical difference between the groups was calculated with one way analysis of variance followed by Neuman– Keuls’ post-test where P < 0.05 was considered significant.
Fig. 2. Representative examples of urothelial ATP release in control and cyclophosphamide (CYP) treated bladders. The preparation was superfused with oxygenated Krebs solution at a rate of 0.5 ml/min. Following a 60-min washout period, 5-min effluents were collected using a fraction collector. After taking two baseline samples, the urothelial surface was exposed at ‘‘"’’ to hypoosmotic Krebs solution for 6 min and then perfused with isoosmotic Krebs solution for another 25 min. The released ATP was expressed as fmol/cm2 based on the calculated urothelial surface area within the Ussing chamber.
indicate that the basal release was not significantly increased after chronic CYP treatment nor was it changed following BTX-A instillation.
3. Results 3.2. Hypoosmotic stimulation evoked release 3.1. Urothelial ATP release 3.1.1. Basal release The average of the first and last two samples in the course of the perfusion was considered as a measure of the basal release of ATP from the urothelial side. In most circumstances, the basal outflow of ATP did not change significantly after hypoosmotic stimulation. However, in a few cases, the basal release showed a decreasing tendency over time. Thus, using only the first two values to calculate basal outflow would overestimate the resting release. The results shown in Fig. 1
Fig. 1. Histogram of basal release of ATP from rat bladder urothelium in control (CTRL), botulinum toxin A (BTX), cyclophosphamide (CYP) and cyclophosphamide + botulinum toxin A (CYP-BTX) treated bladders. The ordinate indicates the release of ATP expressed as fmol/cm2, where the ATP release was calculated on the basis of the bladder area contained within the Ussing chamber. Note that none of the treatments significantly changed the basal release of ATP from bladder urothelium. For number of animals (see Table 1).
As shown in Figs. 2 and 3 animals treated with CYP had a greater urothelial ATP release in response to hypoosmotic stimulation than animals treated with saline alone. In addition, treatment with BTX-A reduced evoked ATP release in CYP treated animals by 69%. However, BTX-A instillation did not significantly alter ATP release from saline treated animals. No significant release of ATP above baseline levels was noted in perfusates from the serosal surface of the bladder in
Fig. 3. Histogram of area under the evoked-ATP release curve in response to hypoosmotic stimulation in saline control animals (CTRL) and animals treated with botulinum toxin A (BTX), cyclophosphamide (CYP), or cyclophosphamide + botulinum toxin A (CYP-BTX). Ordinate indicates the evoked release of ATP expressed as percent of the basal release (see Section 2). Chronic treatment with CYP significantly increased urothelial release of ATP as compared to saline treated animals (*P < 0.05). In addition, pre-treatment with BTX-A significantly reduced the amount of ATP released in CYP treated animals (##P < 0.01) but did not significantly affect ATP release in control animals. For number of animals (see Table 1).
294
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
Table 1 In vivo effect of BTX-A on the frequency of voiding and non-voiding contractions after chronic CYP-induced inflammation
CTRL BTX-A CYP CYP + BTX-A
Voiding contractions (# of contractions/h)
Non-voiding contractions (# of contractions/h)
11.63 1.65 (n = 5) 12.1 2.37 (n = 5) 9.64 1.56 (n = 6) 10.65 4.4 (n = 5)
11.9 3.4 (n = 5) 2.11 0.5*(n = 5) 20.11 3.6* (n = 6) 1.78 0.4### (n = 5)
The numbers in the table represent the frequency of bladder contractions per hour. Note that there was no significant difference between the effect of different treatments with regard to voiding contractions; however, CYP treatment significantly increased the frequency of non-voiding bladder contractions while BTX-A (20 U/ml) instilled into the bladder lumen significantly decreased the frequency of non-voiding bladder contractions in both saline treated and CYP treated animals. n = number of animals per group. * P < 0.05 compared to control group. ### P < 0.001 between CYP and CYP + BTX-A treated group, one way analysis of variance with Neuman–Keuls post-test.
response to hypoosmotic urothelial stimulation in any of the Ussing chamber experiments conducted (data not shown). 3.3. In vivo action of BTX-A on bladder contraction frequency Voiding and non-voiding bladder contractions were affected to differing extents when CYP and/or BTX were applied. As shown in Table 1, CYP induced bladder hyperactivity by significantly increasing the rate of nonvoiding bladder contractions by 69%. However, BTX-A instillation markedly reduced bladder hyperactivity induced by CYP by reducing non-voiding contraction frequency by 91%. In addition, BTX-A also reduced non-voiding bladder contraction frequency in saline treated animals but to a lesser extent (Table 1). However, neither CYP nor BTX-A nor a combination of CYP + BTX-A had any affect on the frequency of voiding bladder contractions.
4. Discussion The purpose of this study was to examine the effects of intravesical BTX-A on afferent responses in a rat model of chronic cystitis focusing, in particular, on the interaction between urothelium and bladder afferent nerve fibers. The primary findings of these experiments are that: (1) urothelial ATP release in response to hypoosmotic stimulation is significantly increased during CYP induced chronic bladder inflammation, (2) evoked urothelial ATP release following CYP induced inflammation is significantly reduced by intravesical BTX-A instillation and (3) altered urothelial release of ATP reflects changes in C-fiber nerve activity, since only the frequency of non-voiding but not voiding bladder contractions was affected. Studies have shown that ATP release is increased in response to bladder or ureteral stretch (Ferguson et al., 1997;
Knight et al., 2002). In this study, we applied hypoosmotic shock to evoke mechanical stress. A number of studies support the theory that ATP released by the urothelium plays an important role in the sensory response to bladder distension. (1) Intravesical administration of ATP induces bladder contractions in unanesthetized rats (Pandita and Andersson, 2002). (2) Unmyelinated c-fiber afferents are located between urothelial cells or adjacent to the basal surface of the urothelium and, thus, are positioned to act as mechanosensory receptors (Sengupta and Gebhart, 1994). (3) Finally, studies have shown a significantly reduced bladder afferent nerve response to stretch in P2X3 deficient mice or following application of ATP antagonists in normal mice (Vlaskovska et al., 2001). Investigations in human and animal models of chronic bladder inflammation imply that plasticity of urothelial transmitter release may play an important role in underlying bladder pathophysiology. For example, Birder et al. (2003) showed that, in urothelial cells from cats with feline interstitial cystitis, ATP release in response to hypoosmotic swelling was significantly greater than in urothelial cells from healthy adult cats. In a similar fashion, urothelium from human patients with interstitial cystitis, a disorder of chronic bladder inflammation, release significantly higher quantities of ATP in response to stretch compared to urothelial cells from control patients (Sun et al., 2001). Moreover, our present study demonstrating that mechanically evoked urothelial release of ATP in rats with CYP induced chemical cystitis is significantly greater than in control animals, provides further proof that alterations in urothelial cell effector function do occur after chronic bladder inflammation. ATP appears to be released from urothelial cells via exocytotic mechanisms in response to mechanical stimulation (Knight et al., 2002; Khera et al., 2004). In our study, while evoked ATP release was significantly increased in CYP treated animals, we also found that ATP release in response to stretch could be significantly inhibited by pretreatment with intravesically applied BTX-A. Prior studies in spinal cord injured urothelium and intestine have shown that release of ATP can be inhibited by pre-treatment with BTX, suggesting that, epithelial ATP release occurs via exocytosis and is mediated by the SNARE complex (van der Wijk et al., 2003; Khera et al., 2004). Our finding that CYP treatment increases evoked release of ATP that was inhibited by intravesical instillation of BTX-A suggests that urothelial SNARE mechanisms may be enhanced after chronic bladder inflammation. While hypoosmotic induced release of ATP was increased in CYP induced chronic bladder inflammation and, conversely, decreased by pre-treatment with intravesical BTX-A, we observed no significant differences in basal outflow of ATP under any of our experimental conditions. The fact that BTX-A had no effect on basal ATP levels suggests that this pool of ATP is not released by exocytotic mechanisms and is consistent with prior investigations in SCI animals.
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
Torres et al. (2002) have previously demonstrated that hydrolysis of ATP is decreased under stressful conditions or conditions of increased nerve activity (Cunha et al., 1996) because of a reduction in ectonucleotidase enzyme activity that metabolizes ATP to adenosine. In this regard, one could postulate that the increase in basal and hypoosmotic evoked release of ATP in CYP treated animals could be a result of increased levels of ATP secondary to decreased metabolism. While we did not directly measure the effects of ATP metabolism in these experiments, the fact that BTX-A treatment significantly reduced hypoosmotic evoked ATP release in CYP animals suggests that the majority of this pool of ATP originates from synaptic vesicles. However, because basal release of ATP was not impacted by BTX-A raises the possibility that increase in basal ATP levels after CYP induced inflammation is the result of non-vesicular ATP release and/or altered hydrolysis of ATP. We utilized cystometry to correlate changes in urothelial ATP release with clinical changes in rat global voiding function. Prior studies have shown that chemical cystitis induces bladder hyperactivity by increasing the sensitivity of c-fiber afferent neurons and by recruiting normally silent afferent fibers (Habler et al., 1990; Yoshimura and de Groat, 1998). We have previously demonstrated in a cyclophosphamide model of chronic bladder inflammation that intravesical BTX-A instillation reduces spinal cord c-fos expression, an indirect measure of bladder afferent nerve activity (Vemulakonda et al., 2005). To evaluate the mechanism by which intravesically applied BTX-A inhibits afferent nerve activity, we assessed the effect of BTX-A on ATP release from the bladder urothelium as a marker and a
295
trigger for afferent nerve activation. We then correlated changes in ATP release with changes in the frequency of voiding and non-voiding bladder contractions (Khera et al., 2004; Vemulakonda et al., 2005). Prior experiments in spinal cord injured rats have demonstrated that non-voiding bladder contractions are mediated by capsaicin-sensitive bladder afferent nerves (Cheng et al., 1995). Our results demonstrated a direct correlation between CYP induced increases in urothelial ATP release and increases in the frequency of non-voiding contractions as well as BTX-A induced reductions in urothelial ATP release and reductions in the frequency of non-voiding bladder contractions. These data strongly suggest that both CYP induced bladder inflammation and BTX-A treatment selectively act on capsaicin-sensitive afferent nerve fibers. Moreover, the fact that changes in frequency of bladder contractions coincided with changes in urothelial ATP release provides evidence of a functional relevance for the pool of ATP evoked by mechanical stimulation (Fig. 4). In contrast, the frequency of voiding contractions, which are predominantly driven by Ad afferent nerve fibers, was not significantly changed after CYP or BTX-A treatment. Given prior investigations documenting an increase in C-fiber activity in conditions of chronic inflammation as well as our prior evidence that BTX is more effective in conditions of greater nerve activity, any effect of BTX-A on Ad afferent nerve fibers may have been masked (Yoshimura and de Groat, 1999; Smith et al., 2003). One might suggest that vesicular release mechanisms do not operate under normal conditions and are activated de novo by inflammation. Alternatively, a more plausible explanation is that vesicular mechanisms are
Fig. 4. Schematic diagram of the afferent innervation of the bladder as well as the hypothesized action of urothelial released ATP on afferent nerve subpopulations. ATP released from the urothelium acts on the pressure and volume receptors located in the suburothelial layer. Under normal conditions, unmyelinated c-fibers are silent and, thus, urothelial-released ATP does not activate c-fiber afferents. During pathologic conditions, such as following chronic bladder inflammation from CYP treatment, c-fiber afferents become responsive to mechanical stimulation and, we hypothesize, become sensitized to the enhanced release of urothelial ATP into suburothelial tissue layers. The end result of C-fiber activation is bladder hyperactivity evidenced on cystometry by the increased frequency of non-voiding contractions.
296
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
enhanced either by increased frequency of nerve stimulation or by inducing pathology where the clinical inhibitory effects of BTX-A now become measurable. Our hypothesis is supported by a recent publication by Boudreault and Grygorczyk (2004) demonstrating that calcium-dependent exocytosis is a major mechanism of cell-swelling induced ATP release from epithelium. We were somewhat puzzled by the significant effect of BTX-A on reducing non-voiding contractions in our control animals. Our control animals were tested 10 days after protamine sulfate instillation and, thus, we cannot discount the possibility that subclinical inflammation may have occurred in our control group even though prior electrophysiological studies have demonstrated restoration of normal urothelial membrane resistance within a few days after protamine instillation (Apodaca et al., 2003). In fact, we have previously shown that testing animals 10 days after protamine sulfate instillation leads to an increase in nonvoiding bladder contraction frequency as well as an increase in spinal cord c-fos levels compared to non-protamine treated controls (Vemulakonda et al., 2005). Moreover, the fact that BTX-A reduced evoked ATP release in control animals by 55%, although not statistically significant, may have played a role in reducing afferent nerve activation and non-voiding bladder contraction frequency. In conclusion, our studies suggest that: (1) CYP induced bladder inflammation induces plasticity of urothelium that leads to an enhanced release of ATP and an increase in afferent nerve activity, (2) BTX-A inhibits the afferent neural response via inhibition of mechanoreceptor mediated release of ATP and (3) mechanically evoked ATP release following chronic bladder inflammation acts predominantly on c-fiber afferents that mediate non-voiding bladder contractions. Our findings open the possibility that intravesical instillation of BTX-A may ameliorate the clinical symptoms of interstitial cystitis such as urinary frequency, urgency and pain.
Acknowledgements We express our sincere gratitude to K. Roger Aoki, Ph.D., for valuable discussions and providing the BTX-A (Botox1, Allergan, Irvine, CA) for our experiments. This work was supported by the NIH grant RO1 DK 069988; The Scott Department of Urology Neurourology Fund; and an Unrestricted Educational Grant from Allergan, Irvine, CA.
References Apodaca, G., Kiss, S., Ruiz, W., Meyers, S., Zeidel, M., Birder, L., 2003. Disruption of bladder epithelium barrier function after spinal cord injury. Am. J. Physiol. 284 (5), F966–F976. Birder, L.A., Barrick, S.R., Roppolo, J.R., Kanai, A.J., de Groat, W.C., Kiss, S., Buffington, C.A., 2003. Feline interstitial cystitis results in mechan-
ical hypersensitivity and altered ATP release from bladder urothelium. Am. J. Physiol., Renal Physiol. 285 (3), F423–F429. Birder, L.A., Nakamura, Y., Kiss, S., Nealen, M.L., Barrick, S., Kanai, A.J., Wang, E., Ruiz, G., de Groat, W.C., Apodaca, G., Watkins, S., Caterina, M.J., 2002. Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1. Nat. Neurosci. 5 (9), 856–860. Boudreault, F., Grygorczyk, R., 2004. Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations. J. Physiol. 561 (Pt. 2), 499–513. Cheng, C.L., Ma, C.P., de Groat, W.C., 1995. Effect of capsaicin on micturition and associated reflexes in chronic spinal rats. Brain Res. 678 (1–2), 40–48. Chuang, Y.C., Yoshimura, N., Huang, C.C., Chiang, P.H., Chancellor, M.B., 2004. Intravesical botulinum toxin a administration produces analgesia against acetic acid induced bladder pain responses in rats. J. Urol. 172 (4 Pt. 1), 1529–1532. Cui, M., Khanijou, S., Rubino, J., Aoki, K.R., 2004. Subcutaneous administration of botulinum toxin A reduces formalin-induced pain. Pain 107, 125. Cunha, R.A., Vizi, E.S., Ribeiro, J.A., Sebastiao, A.M., 1996. Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices. J. Neurochem. 67 (5), 2180–2187. Ferguson, D.R., 1999. Urothelial function. Br. J. Urol. Int. 84 (3), 235– 242. Ferguson, D.R., Kennedy, I., Burton, T.J., 1997. ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes—a possible sensory mechanism? J. Physiol. 505 (Pt. 2), 503–511. Habler, H.J., Janig, W., Koltzenburg, M., 1990. Activation of unmyelinated afferent fibres by mechanical stimuli and inflammation of the urinary bladder in the cat. J. Physiol. 425, 545–562. Habler, H.J., Janig, W., Koltzenburg, M., 1993. Myelinated primary afferents of the sacral spinal cord responding to slow filling and distension of the cat urinary bladder. J. Physiol. 463, 449–460. Khera, M., Somogyi, G.T., Kiss, S., Boone, T.B., Smith, C.P., 2004. Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury. Neurochem. Int. 45 (7), 987–993. Knight, G.E., Bodin, P., de Groat, W.C., Burnstock, G., 2002. ATP is released from guinea pig ureter epithelium on distension. Am. J. Physiol., Renal Physiol. 282 (2), F281–F288. Maggi, C.A., Lecci, A., Santicioli, P., Del Bianco, E., Giuliani, S., 1992. Cyclophosphamide cystitis in rats: involvement of capsaicin-sensitive primary afferents. J. Auton. Nerv. Syst. 38, 201–208. Niku, S.D., Stein, P.C., Scherz, H.C., Parsons, C.L., 1994. A new method for cytodestruction of bladder epithelium using protamine sulfate and urea. J. Urol. 152 (3), 1025–1028. Pandita, R.K., Andersson, K.E., 2002. Intravesical adenosine triphosphate stimulates the micturition reflex in awake, freely moving rats. J. Urol. 168, 1230. Schiavo, G., Santucci, A., DasGupta, B.R., et al., 1993. Botulinum neurotoxins serotypes A and E cleave Snap-25 at distinct COOHterminal peptide bonds. FEBS Lett. 335, 99–103. Sengupta, J.N., Gebhart, G.F., 1994. Mechanosensitive properties of pelvic nerve afferent fibers innervating the urinary bladder of the rat. J. Neurophysiol. 72, 2420–2430. Smith, C.P., Franks, M.E., McNeil, B.K., Ghosh, R., de Groat, W.C., Chancellor, M.B., Somogyi, G.T., 2003. Effect of botulinum toxin A on the autonomic nervous system of the rat lower urinary tract. J. Urol. 169 (5), 1896–1900. Steers, W.D., Tuttle, J.B., 1997. Neurogenic inflammation and nerve growth factor: possible roles in interstitial cystitis. In: Sant, G.R. (Ed.), Interstitial Cystitis. Lippincott-Raven, Philadelphia, pp. 67–75. Sun, Y., Keay, S., De Deyne, P.G., Chai, T.C., 2001. Augmented stretch activated adenosine triphosphate release from bladder uroepithelial cells in patients with interstitial cystitis. J. Urol. 166 (5), 1951–1956. Torres, I.L., Buffon, A., Dantas, G., Furstenau, C.R., Bohmer, A.E., Battastini, A.M., Sarkis, J.J., Dalmaz, C., Ferreira, M.B., 2002. Chronic
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297 stress effects on adenine nucleotide hydrolysis in the blood serum and brain structures of rats. Pharmacol. Biochem. Behav. 74 (1), 181–186. van der Wijk, T., Tomassen, S.F., Houtsmuller, A.B., de Jonge, H.R., Tilly, B.C., 2003. Increased vesicle recycling in response to osmotic cell swelling. Cause and consequence of hypotonicity-provoked ATP release. J. Biol. Chem. 278 (41), 40020–40025. Vemulakonda, V.M., Somogyi, G.T., Kiss, S., Evans, J.L., Khera, M., Boone, T.B., Smith, C.P., 2004. Intravesical botulinum toxin A inhibits the afferent response in the chronically inflamed bladder. J. Urol. 171 (Suppl. 4), 458. Vemulakonda, V.M., Somogyi, G.T., Kiss, S., Salas, N.A., Boone, T.B., Smith, C.P., 2005. Intravesical botulinum toxin A inhibits the afferent response in the chronically inflamed bladder. J. Urol. 173 (2), 621–624. Vizi, E.S., Liang, S.D., Sperlagh, B., Kittel, A., Juranyi, Z., 1997. Studies on the release and extracellular metabolism of endogenous ATP in rat
297
superior cervical ganglion: support for neurotransmitter role of ATP. Neuroscience 79 (3), 893–903. Vizzard, M.A., 2000. Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, 1027–1039. Vlaskovska, M., Kasakov, L., Rong, W., Bodin, P., Bardini, M., Cockayne, D.A., Ford, A.P., Burnstock, G., 2001. P2X3 knock-out mice reveal a major sensory role for urothelially released ATP. J. Neurosci. 21 (15), 5670–5677. Welch, M.J., Purkiss, J.R., Foster, K.A., 2000. Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins. Toxicon 38, 245–258. Yoshimura, N., de Groat, W.C., 1999. Increased excitability of afferent neurons innervating rat urinary bladder after chronic bladder inflammation. J. Neurosci. 19 (11), 644–653.
Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: Effect of botulinum toxin A Christopher P. Smith, Vijaya M. Vemulakonda, Susanna Kiss, Timothy B. Boone, George T. Somogyi * Scott Department of Urology, Baylor College of Medicine, One Baylor Plaza, Alkek N720, Houston, TX 77030, USA Received 7 January 2005; received in revised form 10 April 2005; accepted 11 April 2005 Available online 20 June 2005
Abstract The effects of mechanoreceptor stimulation and subsequent ATP release in cyclophosphamide evoked chronic bladder inflammation was examined to demonstrate: (1) whether inflammation modulates ATP release from bladder urothelium and (2) whether intravesical botulinum toxin A administration inhibits urothelial ATP release, a measure of sensory nerve activation. ATP release was measured from rat bladders in a Ussing chamber, an apparatus that allows one to separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Cystometry was utilized to correlate changes in ATP release with alterations in the frequency of voiding and non-voiding bladder contractions, in vivo measures of bladder afferent activity. The resting urothelial release of ATP was not significantly affected by either cyclophosphamide or botulinum toxin A treatment. However, evoked ATP release following hypoosmotic stimulation was significantly increased (i.e. 94%) in chronic cyclophosphamide treated bladder urothelium compared to control bladders. In addition, botulinum toxin A treatment significantly reduced hypoosmotic shock induced ATP release in cyclophosphamide treated animals by 69%. Cystometry revealed that cyclophosphamide and botulinum toxin A treatments altered non-voiding (i.e. cyclophosphamide increased, botulinum toxin A decreased) but not voiding contraction frequency suggesting that alterations in urothelial ATP release selectively diminished underlying bladder C-fiber nerve activity. Finally, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side implying that its effects were confined to the urothelial side of the bladder preparation. # 2005 Elsevier Ltd. All rights reserved. Keywords: Botulinum toxin; Bladder; Inflammation; Interstitial cystitis; Ussing chamber; ATP; Urothelium
1. Introduction Chronic inflammatory conditions have been shown to lower the pain threshold and increase perceptions of pain. Inflammation in visceral organs causes increased sensitivity to noxious and non-noxious stimulation (Habler et al., 1993). Cyclophosphamide (CYP) induced cystitis, an animal model of chronic bladder inflammation, has been shown to increase voiding frequency and induce bladder hyperactivity both acutely and chronically (Maggi et al., 1992; Vizzard, 2000).
* Corresponding author. Tel.: +1 713 798 3541; fax: +1 713 798 6454. E-mail address: [email protected] (G.T. Somogyi). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.04.021
ATP is known to be a prominent neurotransmitter at both efferent (Vizi et al., 1997) and sensory sites within the peripheral nervous system. Recent findings support the notion that epithelial tissues including urothelium may be involved in bladder sensory mechanisms (Ferguson et al., 1997; Ferguson, 1999; Birder et al., 2002). For example, activation of the urothelium by pressure, stretch or hypoosmotic shock releases ATP from the urothelial cells, both in cell culture and an in vitro system where an Ussing chamber is used to separate the urothelial and serosal sides (Ferguson et al., 1997; Khera et al., 2004). In this context, the increase in ATP release can be used as a marker for activation of the bladder urothelium. Prior investigations have demonstrated that ATP release from cultured urothelial cells is increased in patients suffering from interstitial
292
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
cystitis (IC) (Sun et al., 2001), a bladder disorder characterized by chronic inflammation and pain and thought to result, in part, from alterations in urothelial and afferent nerve function (Steers and Tuttle, 1997). Botulinum toxin A (BTX-A) inhibits neurotransmitter release from nerve terminals by inhibiting the fusion of the intracytoplasmic vesicle with the plasma membrane (Schiavo et al., 1993). We have previously demonstrated that BTX-A inhibits acetylcholine and norepinephrine release from efferent nerve terminals in the lower urinary tract (Smith et al., 2003). However, recent studies suggest that BTX may also exert a considerable inhibitory action on the sensory nervous system. For example, BTX-A has been shown to decrease neuropeptide release from rat dorsal root ganglia (Welch et al., 2000) and inhibit ATP release from the epithelium of intestine as well as the urothelium of neurogenic bladders (Khera et al., 2004). Furthermore, in vivo models of somatic (Cui et al., 2004) and visceral inflammation (Vemulakonda et al., 2005) and irritation (Chuang et al., 2004) support an anti-nociceptive effect of BTX-A. In this study, we evaluated the effect of chronic inflammation on hypoosmotic shock-induced ATP release from bladder urothelium using the previously described modified Ussing chamber model (Khera et al., 2004). Furthermore, we analyzed afferent effects of intravesically instilled BTX-A using ATP release as a marker under these noxious circumstances. Experiments were also conducted utilizing cystometry to correlate the in vitro changes in ATP release with the in vivo effects of chronic bladder inflammation and intravesical BTX-A instillation. A preliminary report of these results has been published (Vemulakonda et al., 2004).
2. Materials and methods 2.1. Preparation Female Sprague–Dawley rats weighing 200–250 g were divided by intravesical instillation and intraperitoneal injection into four groups (n = 5–6 per group) for both in vivo and in vitro experiments. (1) Saline instilled and saline injected (Control). (2). BTX-A instilled and saline injected (BTX). (3) Saline instilled and CYP injected (CYP). (4) BTX-A instilled and CYP injected (CYP + BTX). The effectiveness of chronic CYP treatment as well as the inhibitory effect of BTX-A instillation was evaluated with bladder cystometry by monitoring the frequency of voiding and non-voiding bladder contractions. All animal experiments were carried out in accordance with the National Institutes of Health guideline. 2.2. Experimental protocol On day 0, under isoflurane anesthesia rat bladders were filled with 1 ml of 1% protamine sulfate (Sigma, St. Louis,
MO) transurethrally for 30 min to disrupt the integrity of the urothelial barrier (Niku et al., 1994). The protamine sulfate was drained and the bladder was instilled either with 1 ml of 20 units BTX-A (Botox1, Allergan, Irvine, CA) or saline for 30 min (Khera et al., 2004; Vemulakonda et al., 2005). One unit of Botox1 equals the calculated intraperitoneal LD50 in mice (Botox1 package insert). Chronic cystitis was induced by injecting 150 mg/kg CYP (Sigma) intraperitoneally every third day for a total of three injections while control animals received the same volume of i.p. saline injection. Both the in vivo and in vitro experiments were performed on day 10. 2.2.1. Measurement of in vivo bladder contractions On the tenth day, a suprapubic catheter was placed through the bladder dome under isoflurane anesthesia. After recovery from anesthesia, the animals were restrained (Universal Rodent Restrainer, Harvard Apparatus, Holliston, MA) and the bladder was perfused with saline at a rate of 0.04 ml/min using a syringe pump (KDS, New Hope, PA). Bladder contractions and voided urine were measured with a pressure and force transducer system (WPI, Sarasota, FL), respectively, as described in earlier studies (Vemulakonda et al., 2005). The frequency of non-voiding or voiding bladder contractions (i.e. contractions where urine was expelled) was calculated and expressed as contractions/h. An increase in contraction frequency represented an increase in afferent nerve activity and vice-versa. The frequency of both types of bladder contractions was calculated using the Windaq playback program (WindaqEx, DATAQ, Akron, OH). 2.2.2. Measuring urothelial ATP release After completion of cystometrogram evaluation, animals were sacrificed and bladders were removed and mounted in an Ussing chamber, a tissue bath that consists of two parts separated by the bladder tissue cut opened where one chamber faces the urothelial side and the other chamber faces the serosal side of the bladder (for details see Khera et al., 2004). The serosal and urothelial hemichambers were separately perfused with oxygenated preheated 37 8C Krebs solution (mm/l; NaCl 113, KCl 4.7, CaCl2 1.25, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11.5) at a rate of 0.5 ml/ min with a peristaltic pump (Rainin, Minipuls2, Woburn, MA) for 1 h and 5 min effluents were collected using a fraction collector (Gilson, Middleton, WI). After taking two baseline samples, the urothelial surface was exposed to hypoosmotic Krebs solution (i.e. NaCl concentration was reduced by 40% from 113 to 67.8 mM) for 6 min and then perfused with isoosmotic Krebs solution for another 25 min. A 50 ml aliquot from each 5 min effluent was placed into a luminometer (Turner, Sunnyvale, CA) and the ATP content was measured by using the luciferin-luciferase assay. The luminescence of the collected samples was compared to that of standard ATP and the basal and evoked release was calculated. The average of the first and last samples in the
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
293
course of the perfusion was considered, in most cases, as the basal release of urothelial ATP and was standardized to the bladder surface area (i.e. fmol/cm2). Evoked release of ATP by hypoosmotic stimulation was expressed as area computed from the percent increase over the basal release of ATP. 2.3. Drugs Cyclophosphamide, protamine sulfate, the ATP bioluminescent assay kit and all constituents of Krebs solution were purchased from Sigma. BTX-A (Botox1) was a generous gift from Allergan Inc. (Irvin, CA). 2.4. Statistical analysis Statistical analysis was performed using Prism3 software (Graph Pad, San Diego, CA). The mean and standard error of the mean were calculated for each series of measurements. The statistical difference between the groups was calculated with one way analysis of variance followed by Neuman– Keuls’ post-test where P < 0.05 was considered significant.
Fig. 2. Representative examples of urothelial ATP release in control and cyclophosphamide (CYP) treated bladders. The preparation was superfused with oxygenated Krebs solution at a rate of 0.5 ml/min. Following a 60-min washout period, 5-min effluents were collected using a fraction collector. After taking two baseline samples, the urothelial surface was exposed at ‘‘"’’ to hypoosmotic Krebs solution for 6 min and then perfused with isoosmotic Krebs solution for another 25 min. The released ATP was expressed as fmol/cm2 based on the calculated urothelial surface area within the Ussing chamber.
indicate that the basal release was not significantly increased after chronic CYP treatment nor was it changed following BTX-A instillation.
3. Results 3.2. Hypoosmotic stimulation evoked release 3.1. Urothelial ATP release 3.1.1. Basal release The average of the first and last two samples in the course of the perfusion was considered as a measure of the basal release of ATP from the urothelial side. In most circumstances, the basal outflow of ATP did not change significantly after hypoosmotic stimulation. However, in a few cases, the basal release showed a decreasing tendency over time. Thus, using only the first two values to calculate basal outflow would overestimate the resting release. The results shown in Fig. 1
Fig. 1. Histogram of basal release of ATP from rat bladder urothelium in control (CTRL), botulinum toxin A (BTX), cyclophosphamide (CYP) and cyclophosphamide + botulinum toxin A (CYP-BTX) treated bladders. The ordinate indicates the release of ATP expressed as fmol/cm2, where the ATP release was calculated on the basis of the bladder area contained within the Ussing chamber. Note that none of the treatments significantly changed the basal release of ATP from bladder urothelium. For number of animals (see Table 1).
As shown in Figs. 2 and 3 animals treated with CYP had a greater urothelial ATP release in response to hypoosmotic stimulation than animals treated with saline alone. In addition, treatment with BTX-A reduced evoked ATP release in CYP treated animals by 69%. However, BTX-A instillation did not significantly alter ATP release from saline treated animals. No significant release of ATP above baseline levels was noted in perfusates from the serosal surface of the bladder in
Fig. 3. Histogram of area under the evoked-ATP release curve in response to hypoosmotic stimulation in saline control animals (CTRL) and animals treated with botulinum toxin A (BTX), cyclophosphamide (CYP), or cyclophosphamide + botulinum toxin A (CYP-BTX). Ordinate indicates the evoked release of ATP expressed as percent of the basal release (see Section 2). Chronic treatment with CYP significantly increased urothelial release of ATP as compared to saline treated animals (*P < 0.05). In addition, pre-treatment with BTX-A significantly reduced the amount of ATP released in CYP treated animals (##P < 0.01) but did not significantly affect ATP release in control animals. For number of animals (see Table 1).
294
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
Table 1 In vivo effect of BTX-A on the frequency of voiding and non-voiding contractions after chronic CYP-induced inflammation
CTRL BTX-A CYP CYP + BTX-A
Voiding contractions (# of contractions/h)
Non-voiding contractions (# of contractions/h)
11.63 1.65 (n = 5) 12.1 2.37 (n = 5) 9.64 1.56 (n = 6) 10.65 4.4 (n = 5)
11.9 3.4 (n = 5) 2.11 0.5*(n = 5) 20.11 3.6* (n = 6) 1.78 0.4### (n = 5)
The numbers in the table represent the frequency of bladder contractions per hour. Note that there was no significant difference between the effect of different treatments with regard to voiding contractions; however, CYP treatment significantly increased the frequency of non-voiding bladder contractions while BTX-A (20 U/ml) instilled into the bladder lumen significantly decreased the frequency of non-voiding bladder contractions in both saline treated and CYP treated animals. n = number of animals per group. * P < 0.05 compared to control group. ### P < 0.001 between CYP and CYP + BTX-A treated group, one way analysis of variance with Neuman–Keuls post-test.
response to hypoosmotic urothelial stimulation in any of the Ussing chamber experiments conducted (data not shown). 3.3. In vivo action of BTX-A on bladder contraction frequency Voiding and non-voiding bladder contractions were affected to differing extents when CYP and/or BTX were applied. As shown in Table 1, CYP induced bladder hyperactivity by significantly increasing the rate of nonvoiding bladder contractions by 69%. However, BTX-A instillation markedly reduced bladder hyperactivity induced by CYP by reducing non-voiding contraction frequency by 91%. In addition, BTX-A also reduced non-voiding bladder contraction frequency in saline treated animals but to a lesser extent (Table 1). However, neither CYP nor BTX-A nor a combination of CYP + BTX-A had any affect on the frequency of voiding bladder contractions.
4. Discussion The purpose of this study was to examine the effects of intravesical BTX-A on afferent responses in a rat model of chronic cystitis focusing, in particular, on the interaction between urothelium and bladder afferent nerve fibers. The primary findings of these experiments are that: (1) urothelial ATP release in response to hypoosmotic stimulation is significantly increased during CYP induced chronic bladder inflammation, (2) evoked urothelial ATP release following CYP induced inflammation is significantly reduced by intravesical BTX-A instillation and (3) altered urothelial release of ATP reflects changes in C-fiber nerve activity, since only the frequency of non-voiding but not voiding bladder contractions was affected. Studies have shown that ATP release is increased in response to bladder or ureteral stretch (Ferguson et al., 1997;
Knight et al., 2002). In this study, we applied hypoosmotic shock to evoke mechanical stress. A number of studies support the theory that ATP released by the urothelium plays an important role in the sensory response to bladder distension. (1) Intravesical administration of ATP induces bladder contractions in unanesthetized rats (Pandita and Andersson, 2002). (2) Unmyelinated c-fiber afferents are located between urothelial cells or adjacent to the basal surface of the urothelium and, thus, are positioned to act as mechanosensory receptors (Sengupta and Gebhart, 1994). (3) Finally, studies have shown a significantly reduced bladder afferent nerve response to stretch in P2X3 deficient mice or following application of ATP antagonists in normal mice (Vlaskovska et al., 2001). Investigations in human and animal models of chronic bladder inflammation imply that plasticity of urothelial transmitter release may play an important role in underlying bladder pathophysiology. For example, Birder et al. (2003) showed that, in urothelial cells from cats with feline interstitial cystitis, ATP release in response to hypoosmotic swelling was significantly greater than in urothelial cells from healthy adult cats. In a similar fashion, urothelium from human patients with interstitial cystitis, a disorder of chronic bladder inflammation, release significantly higher quantities of ATP in response to stretch compared to urothelial cells from control patients (Sun et al., 2001). Moreover, our present study demonstrating that mechanically evoked urothelial release of ATP in rats with CYP induced chemical cystitis is significantly greater than in control animals, provides further proof that alterations in urothelial cell effector function do occur after chronic bladder inflammation. ATP appears to be released from urothelial cells via exocytotic mechanisms in response to mechanical stimulation (Knight et al., 2002; Khera et al., 2004). In our study, while evoked ATP release was significantly increased in CYP treated animals, we also found that ATP release in response to stretch could be significantly inhibited by pretreatment with intravesically applied BTX-A. Prior studies in spinal cord injured urothelium and intestine have shown that release of ATP can be inhibited by pre-treatment with BTX, suggesting that, epithelial ATP release occurs via exocytosis and is mediated by the SNARE complex (van der Wijk et al., 2003; Khera et al., 2004). Our finding that CYP treatment increases evoked release of ATP that was inhibited by intravesical instillation of BTX-A suggests that urothelial SNARE mechanisms may be enhanced after chronic bladder inflammation. While hypoosmotic induced release of ATP was increased in CYP induced chronic bladder inflammation and, conversely, decreased by pre-treatment with intravesical BTX-A, we observed no significant differences in basal outflow of ATP under any of our experimental conditions. The fact that BTX-A had no effect on basal ATP levels suggests that this pool of ATP is not released by exocytotic mechanisms and is consistent with prior investigations in SCI animals.
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
Torres et al. (2002) have previously demonstrated that hydrolysis of ATP is decreased under stressful conditions or conditions of increased nerve activity (Cunha et al., 1996) because of a reduction in ectonucleotidase enzyme activity that metabolizes ATP to adenosine. In this regard, one could postulate that the increase in basal and hypoosmotic evoked release of ATP in CYP treated animals could be a result of increased levels of ATP secondary to decreased metabolism. While we did not directly measure the effects of ATP metabolism in these experiments, the fact that BTX-A treatment significantly reduced hypoosmotic evoked ATP release in CYP animals suggests that the majority of this pool of ATP originates from synaptic vesicles. However, because basal release of ATP was not impacted by BTX-A raises the possibility that increase in basal ATP levels after CYP induced inflammation is the result of non-vesicular ATP release and/or altered hydrolysis of ATP. We utilized cystometry to correlate changes in urothelial ATP release with clinical changes in rat global voiding function. Prior studies have shown that chemical cystitis induces bladder hyperactivity by increasing the sensitivity of c-fiber afferent neurons and by recruiting normally silent afferent fibers (Habler et al., 1990; Yoshimura and de Groat, 1998). We have previously demonstrated in a cyclophosphamide model of chronic bladder inflammation that intravesical BTX-A instillation reduces spinal cord c-fos expression, an indirect measure of bladder afferent nerve activity (Vemulakonda et al., 2005). To evaluate the mechanism by which intravesically applied BTX-A inhibits afferent nerve activity, we assessed the effect of BTX-A on ATP release from the bladder urothelium as a marker and a
295
trigger for afferent nerve activation. We then correlated changes in ATP release with changes in the frequency of voiding and non-voiding bladder contractions (Khera et al., 2004; Vemulakonda et al., 2005). Prior experiments in spinal cord injured rats have demonstrated that non-voiding bladder contractions are mediated by capsaicin-sensitive bladder afferent nerves (Cheng et al., 1995). Our results demonstrated a direct correlation between CYP induced increases in urothelial ATP release and increases in the frequency of non-voiding contractions as well as BTX-A induced reductions in urothelial ATP release and reductions in the frequency of non-voiding bladder contractions. These data strongly suggest that both CYP induced bladder inflammation and BTX-A treatment selectively act on capsaicin-sensitive afferent nerve fibers. Moreover, the fact that changes in frequency of bladder contractions coincided with changes in urothelial ATP release provides evidence of a functional relevance for the pool of ATP evoked by mechanical stimulation (Fig. 4). In contrast, the frequency of voiding contractions, which are predominantly driven by Ad afferent nerve fibers, was not significantly changed after CYP or BTX-A treatment. Given prior investigations documenting an increase in C-fiber activity in conditions of chronic inflammation as well as our prior evidence that BTX is more effective in conditions of greater nerve activity, any effect of BTX-A on Ad afferent nerve fibers may have been masked (Yoshimura and de Groat, 1999; Smith et al., 2003). One might suggest that vesicular release mechanisms do not operate under normal conditions and are activated de novo by inflammation. Alternatively, a more plausible explanation is that vesicular mechanisms are
Fig. 4. Schematic diagram of the afferent innervation of the bladder as well as the hypothesized action of urothelial released ATP on afferent nerve subpopulations. ATP released from the urothelium acts on the pressure and volume receptors located in the suburothelial layer. Under normal conditions, unmyelinated c-fibers are silent and, thus, urothelial-released ATP does not activate c-fiber afferents. During pathologic conditions, such as following chronic bladder inflammation from CYP treatment, c-fiber afferents become responsive to mechanical stimulation and, we hypothesize, become sensitized to the enhanced release of urothelial ATP into suburothelial tissue layers. The end result of C-fiber activation is bladder hyperactivity evidenced on cystometry by the increased frequency of non-voiding contractions.
296
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297
enhanced either by increased frequency of nerve stimulation or by inducing pathology where the clinical inhibitory effects of BTX-A now become measurable. Our hypothesis is supported by a recent publication by Boudreault and Grygorczyk (2004) demonstrating that calcium-dependent exocytosis is a major mechanism of cell-swelling induced ATP release from epithelium. We were somewhat puzzled by the significant effect of BTX-A on reducing non-voiding contractions in our control animals. Our control animals were tested 10 days after protamine sulfate instillation and, thus, we cannot discount the possibility that subclinical inflammation may have occurred in our control group even though prior electrophysiological studies have demonstrated restoration of normal urothelial membrane resistance within a few days after protamine instillation (Apodaca et al., 2003). In fact, we have previously shown that testing animals 10 days after protamine sulfate instillation leads to an increase in nonvoiding bladder contraction frequency as well as an increase in spinal cord c-fos levels compared to non-protamine treated controls (Vemulakonda et al., 2005). Moreover, the fact that BTX-A reduced evoked ATP release in control animals by 55%, although not statistically significant, may have played a role in reducing afferent nerve activation and non-voiding bladder contraction frequency. In conclusion, our studies suggest that: (1) CYP induced bladder inflammation induces plasticity of urothelium that leads to an enhanced release of ATP and an increase in afferent nerve activity, (2) BTX-A inhibits the afferent neural response via inhibition of mechanoreceptor mediated release of ATP and (3) mechanically evoked ATP release following chronic bladder inflammation acts predominantly on c-fiber afferents that mediate non-voiding bladder contractions. Our findings open the possibility that intravesical instillation of BTX-A may ameliorate the clinical symptoms of interstitial cystitis such as urinary frequency, urgency and pain.
Acknowledgements We express our sincere gratitude to K. Roger Aoki, Ph.D., for valuable discussions and providing the BTX-A (Botox1, Allergan, Irvine, CA) for our experiments. This work was supported by the NIH grant RO1 DK 069988; The Scott Department of Urology Neurourology Fund; and an Unrestricted Educational Grant from Allergan, Irvine, CA.
References Apodaca, G., Kiss, S., Ruiz, W., Meyers, S., Zeidel, M., Birder, L., 2003. Disruption of bladder epithelium barrier function after spinal cord injury. Am. J. Physiol. 284 (5), F966–F976. Birder, L.A., Barrick, S.R., Roppolo, J.R., Kanai, A.J., de Groat, W.C., Kiss, S., Buffington, C.A., 2003. Feline interstitial cystitis results in mechan-
ical hypersensitivity and altered ATP release from bladder urothelium. Am. J. Physiol., Renal Physiol. 285 (3), F423–F429. Birder, L.A., Nakamura, Y., Kiss, S., Nealen, M.L., Barrick, S., Kanai, A.J., Wang, E., Ruiz, G., de Groat, W.C., Apodaca, G., Watkins, S., Caterina, M.J., 2002. Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1. Nat. Neurosci. 5 (9), 856–860. Boudreault, F., Grygorczyk, R., 2004. Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations. J. Physiol. 561 (Pt. 2), 499–513. Cheng, C.L., Ma, C.P., de Groat, W.C., 1995. Effect of capsaicin on micturition and associated reflexes in chronic spinal rats. Brain Res. 678 (1–2), 40–48. Chuang, Y.C., Yoshimura, N., Huang, C.C., Chiang, P.H., Chancellor, M.B., 2004. Intravesical botulinum toxin a administration produces analgesia against acetic acid induced bladder pain responses in rats. J. Urol. 172 (4 Pt. 1), 1529–1532. Cui, M., Khanijou, S., Rubino, J., Aoki, K.R., 2004. Subcutaneous administration of botulinum toxin A reduces formalin-induced pain. Pain 107, 125. Cunha, R.A., Vizi, E.S., Ribeiro, J.A., Sebastiao, A.M., 1996. Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices. J. Neurochem. 67 (5), 2180–2187. Ferguson, D.R., 1999. Urothelial function. Br. J. Urol. Int. 84 (3), 235– 242. Ferguson, D.R., Kennedy, I., Burton, T.J., 1997. ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes—a possible sensory mechanism? J. Physiol. 505 (Pt. 2), 503–511. Habler, H.J., Janig, W., Koltzenburg, M., 1990. Activation of unmyelinated afferent fibres by mechanical stimuli and inflammation of the urinary bladder in the cat. J. Physiol. 425, 545–562. Habler, H.J., Janig, W., Koltzenburg, M., 1993. Myelinated primary afferents of the sacral spinal cord responding to slow filling and distension of the cat urinary bladder. J. Physiol. 463, 449–460. Khera, M., Somogyi, G.T., Kiss, S., Boone, T.B., Smith, C.P., 2004. Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury. Neurochem. Int. 45 (7), 987–993. Knight, G.E., Bodin, P., de Groat, W.C., Burnstock, G., 2002. ATP is released from guinea pig ureter epithelium on distension. Am. J. Physiol., Renal Physiol. 282 (2), F281–F288. Maggi, C.A., Lecci, A., Santicioli, P., Del Bianco, E., Giuliani, S., 1992. Cyclophosphamide cystitis in rats: involvement of capsaicin-sensitive primary afferents. J. Auton. Nerv. Syst. 38, 201–208. Niku, S.D., Stein, P.C., Scherz, H.C., Parsons, C.L., 1994. A new method for cytodestruction of bladder epithelium using protamine sulfate and urea. J. Urol. 152 (3), 1025–1028. Pandita, R.K., Andersson, K.E., 2002. Intravesical adenosine triphosphate stimulates the micturition reflex in awake, freely moving rats. J. Urol. 168, 1230. Schiavo, G., Santucci, A., DasGupta, B.R., et al., 1993. Botulinum neurotoxins serotypes A and E cleave Snap-25 at distinct COOHterminal peptide bonds. FEBS Lett. 335, 99–103. Sengupta, J.N., Gebhart, G.F., 1994. Mechanosensitive properties of pelvic nerve afferent fibers innervating the urinary bladder of the rat. J. Neurophysiol. 72, 2420–2430. Smith, C.P., Franks, M.E., McNeil, B.K., Ghosh, R., de Groat, W.C., Chancellor, M.B., Somogyi, G.T., 2003. Effect of botulinum toxin A on the autonomic nervous system of the rat lower urinary tract. J. Urol. 169 (5), 1896–1900. Steers, W.D., Tuttle, J.B., 1997. Neurogenic inflammation and nerve growth factor: possible roles in interstitial cystitis. In: Sant, G.R. (Ed.), Interstitial Cystitis. Lippincott-Raven, Philadelphia, pp. 67–75. Sun, Y., Keay, S., De Deyne, P.G., Chai, T.C., 2001. Augmented stretch activated adenosine triphosphate release from bladder uroepithelial cells in patients with interstitial cystitis. J. Urol. 166 (5), 1951–1956. Torres, I.L., Buffon, A., Dantas, G., Furstenau, C.R., Bohmer, A.E., Battastini, A.M., Sarkis, J.J., Dalmaz, C., Ferreira, M.B., 2002. Chronic
C.P. Smith et al. / Neurochemistry International 47 (2005) 291–297 stress effects on adenine nucleotide hydrolysis in the blood serum and brain structures of rats. Pharmacol. Biochem. Behav. 74 (1), 181–186. van der Wijk, T., Tomassen, S.F., Houtsmuller, A.B., de Jonge, H.R., Tilly, B.C., 2003. Increased vesicle recycling in response to osmotic cell swelling. Cause and consequence of hypotonicity-provoked ATP release. J. Biol. Chem. 278 (41), 40020–40025. Vemulakonda, V.M., Somogyi, G.T., Kiss, S., Evans, J.L., Khera, M., Boone, T.B., Smith, C.P., 2004. Intravesical botulinum toxin A inhibits the afferent response in the chronically inflamed bladder. J. Urol. 171 (Suppl. 4), 458. Vemulakonda, V.M., Somogyi, G.T., Kiss, S., Salas, N.A., Boone, T.B., Smith, C.P., 2005. Intravesical botulinum toxin A inhibits the afferent response in the chronically inflamed bladder. J. Urol. 173 (2), 621–624. Vizi, E.S., Liang, S.D., Sperlagh, B., Kittel, A., Juranyi, Z., 1997. Studies on the release and extracellular metabolism of endogenous ATP in rat
297
superior cervical ganglion: support for neurotransmitter role of ATP. Neuroscience 79 (3), 893–903. Vizzard, M.A., 2000. Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, 1027–1039. Vlaskovska, M., Kasakov, L., Rong, W., Bodin, P., Bardini, M., Cockayne, D.A., Ford, A.P., Burnstock, G., 2001. P2X3 knock-out mice reveal a major sensory role for urothelially released ATP. J. Neurosci. 21 (15), 5670–5677. Welch, M.J., Purkiss, J.R., Foster, K.A., 2000. Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins. Toxicon 38, 245–258. Yoshimura, N., de Groat, W.C., 1999. Increased excitability of afferent neurons innervating rat urinary bladder after chronic bladder inflammation. J. Neurosci. 19 (11), 644–653.