Acid-sensing ion channels (ASIC) are a class of ion-channels that are classically expressed in the neurones to sense extracellular acidification and pain. These molecules are also found in a variety of peripheral tissues as a sensory molecule. Recently several subtypes of ASICs have been identified in the urinary bladders, in particular ASIC3 which is preferentially expressed in the urothelium and has been suggested to have important pathological implications(1). However, the functional role of these channels in the bladder is poorly understood, in particular in native tissue. This study used a recently identified ASIC3-specific activator 2-guanidine-4-methylquinazoline (2GMQ) (2;3) to examine the role of ASIC3 channels in urothelium-mediated smooth muscle contraction and ATP release. Guinea-pigs (male Dunkin-Hartley 450-600g) were sacrificed with schedule-1 procedure and urothelium-intact smooth muscle strips and urothelium sheets were isolated from the urinary bladders. The preparation was superfused in a HEPES-buffered Tyrode’s solution and isometric tension was recorded with a tension-transducer via a bridge-amplifier. ATP was measured by sampling the superfusate adjacent to the urothelium and using a luciferin-luciferase assay. Spontaneous contractions were consistently observed in urothelium-intact smooth muscle strips (µN/mg tissue, median (25%-75% range): 734 (272-1966), n=10) which were absent in urothelium-denuded smooth muscle strips. 2GMQ (100µM-500µM) augmented the peak contractile force in urothelium-attached muscle strips (net increase, µN/mg tissue, median (25%-75% range): 100µM 2GMQ: 707 (414 – 1981), n=5, p<0.05, paired Wilcoxon signed-rank test); 500µM 2GMQ: 320 (91-3601), n=5, p<0.05). ATP release was detected from the urothelium attached muscle strips (pmoles/g tissuse/min, median (25%-75% range): 15 (7-90), n=20) and a greater ATP release was generated from the urothelium sheets (182 (23-286), n=20, p<0.05). Application of 2GMQ (10µM-500µM) enhanced ATP release in the urothelium-intact smooth muscle (% control, median (25%-75% range); 10µM 2GMQ: 173 (116 – 276); n=8, p<0.05); 100µM 2GMQ: 250 (177 – 739); n=7, p<0.05; 500µM 2GMQ: 178 (131-270); n=8, p<0.05). Consistently, 2GMQ also increased ATP release from the urothelium sheets (10µM 2GMQ:163 (116-246), n=8, p<0.05); 100µM 2GMQ: 173 (140-296), n=7, p<0.05); 500µM 2GMQ: 165 (135-294), n=5, p<0.05). These data provide the first evidence that ASIC3 channels enhance urothelium-mediated smooth muscle contractions and stimulate ATP release in the bladder wall, mainly attributed to a release from the urothelium. The latter may exert an autocrine/paracrine effect on both motor and sensory function in the urinary bladder.