Neurourology and Urodynamics 30:163–168 (2011) Effects of Intravesical Instillation of ATP on Rat Bladder Primary Afferent Activity and Its Relationship With Capsaicin-Sensitivity Naoki Aizawa, 1,2 Yasuhiko Igawa, 1, ∗ Karl-Erik Andersson, 3 Kazuyoshi Iijima, 2 Osamu Nishizawa, 1 and Jean-Jacques Wyndaele 2 1 Department of Urology, Shinshu University Graduate School of Medicine, Matsumoto, Japan 2 Department of Urology, Faculty of Medicine, University of Antwerp, Antwerp, Belgium 3 Wake Forest Institute for Regenerative Medicine, Wake Forest University, Winston Salem, North Carolina Aims: Previous studies have suggested that ATP has a role in mechano-afferent transduction, at least partly mediated by nerves other than capsaicin (Cap)-sensitive nerves. We investigated the direct effect of ATP on single fiber activity (SFA) of the primary bladder afferent nerves and its relationship with Cap-sensitivity. Methods: Female Sprague--Dawley rats were used. On the basis of conduction velocities, SFA was grouped as A- or C-fibers. First, SFA and intravesical pressure were measured during filling as the baseline. Then, Cap (10 −5 M) was instilled intravesically and the SFA response was monitored. To desensitize Cap-sensitive nerves, resiniferatoxin (RTX) (10 −6 M) was administered intravesically, and then Cap was again administered to confirm the desensitization effect. Thereafter, ATP (10 −3 M) or its vehicle was instilled intravesically and another filling cycles recorded. Results: Thirty-two single afferent fibers were discriminated. A-fibers did not respond to intravesical instillation of Cap, RTX or ATP. Based on Cap-sensitivity, C-fibers could be divided into two subtypes: Cap-sensitive (n = 8) and Cap-insensitive (n = 16). In the Cap-sensitive C-fibers, the response to bladder filling mostly disappeared after RTX desensitization, no matter if the bladder was filled with vehicle or ATP. On the other hand, in the Cap-insensitive C-fibers, even after RTX-treatment, the response was preserved and more enhanced when the bladder was filled with ATP compared to vehicle. Conclusions: Mechanosensitive bladder afferents can be classified as (1) A-fibers, (2) Cap-insensitive, and (3) Cap-sensitive C-fibers. The activation of the bladder afferents induced by intravesical application of ATP is mediated mainly through Cap-insensitive C-fibers. Neurourol. Urodyn. 30:163–168, 2011. © 2010 Wiley-Liss, Inc. Key words: adenosine triphosphate; afferent nerves; capsaicin; rats; urinary bladder INTRODUCTION Primary bladder afferents are generally classified as myeli- nated A-fibers and unmyelinated C-fibers. In the bladder, A-fibers are located primarily within the detrusor smooth mus- cle layer, and respond mainly to detrusor stretching during the phase of bladder filling. They convey sensations of fullness. C- fibers are more widespread, and can be found not only in the detrusor but also in the lamina propria, and often directly adja- cent to the urothelial cells themselves. 1--4 In the rat bladder, both A- and C-fibers include mechanoreceptors, chemoreceptors, and unexcited units. The properties of mechanoreceptors with C-fibers were indistinguishable from those with A-fibers. 5--7 Transient receptor potential vanilloid 1 (TRPV1) receptors are expressed on urothelial cells, but also on bladder primary afferent fibers running suburothelially. These receptors can be activated via stimulation by capsaicin (Cap) or resinifera- toxin (RTX), 8 directly exciting the nerves. However, Cap and RTX are known also to stimulate the release of ATP from the urothelium. ATP can also be released in response to stretch stimuli, such as bladder distension, and is believed to stimu- late P2X 3 purinoceptors on a subpopulation of pelvic afferent nerves 9,10 running in the suburothelial layer of human and rat bladders. 11,12 In a previous study, it was suggested that increased extra- cellular ATP has a role in mechano-afferent transduction in the rat bladder by facilitating the micturition reflex via stimula- tion of nerves other than Cap-sensitive afferents. 13 In this study, we tested whether this is the case by investigating the direct effect of intravesical ATP on A- and C-fiber activities and by determining its relationship with Cap-sensitivity. MATERIALS AND METHODS Animals A total of 25 adult female Sprague--Dawley rats weighing 200--300g was used. The rats were maintained under standard laboratory conditions with a 12:12 h light/dark cycle, and free access to food pellets and tap water. The rats were anesthetized with urethane (0.9g/kg intraperitoneally). Body temperature Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: Ministry of Education, Culture, Sport, Science and Technol- ogy of the Japanese Government; Contract grant number: 20591875. Conflicts of interest: none. Lori Birder led the review process. ∗ Correspondence to: Yasuhiko Igawa M.D., Ph.D., Department of Urology, Shinshu University Graduate School of Medicine, 3-1-1, Asahi, Matsumoto 390-8621, Japan. E-mail: yxigawa@shinshu-u.ac.jp Received 10 October 2009; Accepted 25 March 2010 Published online 21 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/nau.20940 © 2010 Wiley-Liss, Inc.