What's known on the subject? and What does the study add? ATP and acetylcholine (ACh) are released from the urothelium or from cultured urothelial cells in response to mechanical stretching or perturbations that mimic stretching, such as exposure to hypotonic solutions. Limited evidence suggests that ATP and ACh may interact, as the muscarinic agonist carbachol evokes ATP release from cultured urothelial cells. Our aim was to determine whether antimuscarinics, shown previously to reduce sensory nerve firing during bladder filling, might function by reducing mucosal ATP release. By stretching mucosa strips and measuring superfusate ATP using a luciferin–luciferase assay, we showed that muscarinic receptor antagonism (4-DAMP, 10 nM or methoctramine, 1 µM) inhibits stretching-evoked ATP release, suggesting anticholinergic agents used to treat human lower urinary tract pathologies act on urothelial muscarinic receptors. We also showed that alteration of resting mucosal tension is the key determinant of ATP release, as ATP is released from the mucosa in response to relaxation, as well to stretching. OBJECTIVE To determine whether muscarinic receptor antagonism affects stretching-induced release of ATP. MATERIALS AND METHODS Mucosal strips, dissected from guinea pig (male, 450g; n= 10) urinary bladders, were placed in horizontal organ baths and superfused with Ca2+-free Tyrode's solution. Superfusate samples were taken pre- and post- intervention (rapid stretching or relaxation) and ATP concentration was quantified using a luciferin–luciferase assay. The effect of muscarinic acetylcholine receptor antagonism on ATP release was assessed by addition of methoctramine (1 µM) and 4-DAMP (10 nM). RESULTS Rapid stretching (0 to 13.3 ± 1.2 mN; no. strips = 20) increased ATP in the superfusate to a median threefold increase over basal levels. After a period of equilibration, tension in the mucosal strips relaxed until it had reached a new steady-state after 60 min and stretching was repeated. In the presence of 4-DAMP (10 nM) or methoctramine (1 µM), ATP concentrations after stretching reduced to 61% or 20%, respectively. By contrast, ATP concentrations in mucosa-matched controls, perfused with vehicle, increased in response to stretching by 391% and 1500%, respectively. Rapid relaxation also stimulated ATP release. This release did not appear to be sensitive to 4-DAMP or methoctramine. CONCLUSIONS An alteration of resting mucosal tension is the key determinant of ATP release, as ATP is released from the mucosa in response to both stretching and relaxation. Muscarinic receptor antagonism inhibits stretching-evoked ATP release from bladder mucosa, suggesting that anticholinergic agents used to treat human lower urinary tract pathologies act on urothelial muscarinic receptors.