264 RAT AND HUMAN BLADDER UROTHELIAL CELLS HAVE DECREASED ATP-EVOKED INCREASE IN INTRACELLULAR CALCIUM WITH KNOCKDOWN OF P2Y2 RECEPTOR

Abstract

INTRODUCTION AND OBJECTIVES: Urothelium has been recognised as an active sensory structure and pivotal to the signal transduction is ATP release from the urothelium. However, the functional pathways for ATP release mediated by the receptors identified in the urothelium are poorly understood and its paracrine action has never been characterised. This study used native urothelium to test the hypothesis that major urothelial receptors exert significant control over ATP release which further elicits a paracrine effect. METHODS: Human bladder mucosal samples were obtained by cystoscopy with ethics approval and patient consent. Guinea-pig (2-3 month old) urothelial sheets and muscle strips were isolated by blunt dissection. Urothelial cells were freshly dissociated from the urothelial sheets. All preparations were superfused in a physiological saline. A tension transducer was used to record contractions. ATP was measured by sampling the superfusate adjacent to the urothelium and using a luciferin-luciferase assay. Intracellular Ca2 was measured in urothelial cells with Fura-2. RESULTS: Substantial intrinsic ATP release was observed in human (369 97 pmoles/g tissue, n 9 subjects) and guinea-pig urothelium (563 78 pmoles/g, n 46 bladders). P2Y specific agonist UTP increased ATP release (human: 308 34% of control, n 5, p 0.01; guinea-pig: 268 61%, n 11, p 0.01). P2X agonist , methylene ATP had no effect. These effects were mirrored by UTPevoked intracellular Ca rise in urothelial cells. Muscarinic activator carbachol also generated ATP release (human: 512 121% of control, n 7, p 0.05; guinea-pig: 195 23%, n 38, p 0.01). This effect was mimicked by M2 preferential agonist oxotremorine and antagonised by methoctramine (M2), but not by 4-DAMP (M3). Carbachol produced little effect on intracellular Ca but generated mucosal contractions that correlated with ATP release. Urothelium-attached but not urothelium-denuded smooth muscle exhibited intrinsic contractile activities that were augmented by sub-threshold concentrations of carbachol which had little effect on the smooth muscle; these activities were attenuated by desensitizing P2X receptors on the smooth muscle. CONCLUSIONS: Purinergic and muscarinic neurotransmitters exert significant control over urothelial ATP release, mediated by P2Y and M2 receptors. Released ATP produces a paracrine effect on the underlying structures. Whilst intracellular Ca rise may contribute to ATP release via purinergic receptors, mucosal contraction is more likely to underlie ATP release by muscarinic receptors.