Neurourology and Urodynamics 29:789–796 (2010) Maturation of Stretch-Induced Contractile Activity and Its Muscarinic Regulation in Isolated Whole Bladder Strips From Rat Thomas Gevaert, 1 * Grzegorz Owsianik, 2 Graham Hutchings, 1 Wouter Everaerts, 1 Bernd Nilius, 2 and Dirk De Ridder 1 1 Department of Urology, University Hospitals Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium 2 Department of Mol Cell Biology, Division of Physiology, Laboratory of Ion Channel Research, Katholieke Universiteit Leuven, Leuven, Belgium Aim: Besides the establishment of neural reflex pathways, developmental changes in local bladder properties probably also contribute to the onset of mature voiding reflexes. Here we explored the behavior of stretch-induced contractile activity (SIC) and its muscarinic regulation in neonatal and adult rat bladders. Methods: SIC was studied in bladder strips from D0, D7, D28 rat pups and adult rats (15 weeks). The responses to a non-selective [Carbachol (CA), 10 8.5 –10 6 M] and an M 2 -selective muscarinic agonist [arecaidine but-2-ynyl ester tosylate (ABET), 10 9.5 –10 7 M] were studied. The expression of M 2 and M 3 mRNA was investigated using quantitative PCR in whole bladders. The response of SIC to KCl (50 mM) and to the non-adrenergic non-cholinergic (NANC) drugs Substance P (1 mM) and a,b-Methyleneadenosine 5 0 -triphosphate lithium salt (MATP) (1 mM) were also studied. Results: We found evidence for an enhanced response to the muscarinic agonists CA and ABET in neonatal bladders. This might be due to the onset of a direct contractile role for M 2 , given the moderate M 2 -properties of ABET and the absence of ABET-effects on adult bladder strips. Further data showed an increased expression of both M 2 and M 3 at the mRNA level and a changed response of SIC to NANC drugs in neonatal bladder. Conclusions: This study reveals a changed response of SIC to muscarinic and NANC drugs in neonatal rat bladder together with changes at the muscarinic mRNA level, which might all contribute to a better insight in the role of SIC in the onset of mature voiding. Neurourol. Urodynam. 29:789–796, 2010. ß 2010 Wiley-Liss, Inc. Key words: bladder strips; muscarinic; M 2 ;M 3 ; neonatal bladder INTRODUCTION During the first 18 months of life healthy human infants exhibit low bladder capacity, high voiding pressure and frequency, and interrupted voiding. 1 Such infantile voiding gradually evolves toward normal voiding, with voluntary control occurring between 3 and 5 years of age. In several other animal species voiding also undergoes obvious changes during infancy. In rat, neonatal voiding is mediated by a somato-bladder spinal reflex pathway, which is activated when the mother licks the perineum of the neonate. 2,3 Further in the postnatal development (after 10–15 days 3 ) this primitive reflex is replaced by supraspinal mechanisms, which mediate the mature afferent-efferent control of bladder contractility. 2,3 Most research to clarify the mechanisms underlying the onset of mature voiding has focussed on changes in bladder innervation. 2,3 More recently however, contractile and phar- macological differences in isolated neonatal bladder tissue have been reported, which might also be important in the evolution toward mature voiding. Stretch-induced con- tractile activity (SIC) in bladder strips 4,5 has been reported to undergo an evolution from high-amplitude low-frequency contractions in the neonatal period to low-amplitude high- frequency contractions in adults. Pharmacologically the responses to KCl, 6,7 Carbachol (CA), 7 ATP, 6,7 and Substance P 6 were significantly increased in neonatal bladders from differ- ent species. Further reports have been made on age-dependent evolutions in endothelin receptors, 8 neuropeptide Y- and CGRP-related immunoreactive nerve fibres, 9 purinoreceptor expression 10 and calcium-handling in excitation-contraction coupling. 11 (For reviews on age-dependent evolutions in autonomic bladder function, see Refs. 12 and 13). It is thus getting clear that isolated neonatal bladders have particular contractile and pharmacological properties. How- ever, as described by others, the underlying mechanisms are far from being understood. 13 Here we want to add further data to the characterization of SIC in neonatal rat bladder, with particular attention for evolutions in the muscarinic system. We hypothesise that the M 2 -mediated contractile effects are enhanced in neonatal bladder. We studied bladders from four age groups: newborn rat pups (Day 0, D0), neonatal pups (D7), young rats (D28), and adult rats (15 weeks). We chose these age-groups because of specific evolutions in voiding behavior at these time points: 3 voiding induced by maternal licking Conflicts of interest: none. Karl-Erik Andersson led the review process. This article includes Supplementary Material available via the Internet at http:// www.interscience.wiley.com/jpages/0733-2467/suppmat. Grant sponsor: Human Frontiers Science Programme (HFSP); Grant number: RGP 32/2004; Grant sponsor: Belgian Federal Government; Grant sponsor: Flemish Government; Grant sponsor: Onderzoeksraad KU Leuven; Grant numbers: GOA 2004/07, F.W.O. G.0136.00, F.W.O. G.0172.03; Grant sponsor: WOMS; Grant sponsor: Charcot Foundation. *Correspondence to: Thomas Gevaert, M.D., Ph.D., Department of Urology, University Hospitals Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. E-mail: thomas.gevaert@med.kuleuven.ac.be Received 24 September 2007; Accepted 21 November 2007 Published online 15 June 2010 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/nau.20553 ß 2010 Wiley-Liss, Inc.