Muscarinic receptors (mAChRs) have been identified in the urothelium, a tissue that may be involved in bladder sensory mechanisms. This study investigates the expression and function of mAChRs using cultured urothelial cells from the rat. RT-PCR established the expression of all five mAChR subtypes. Muscarinic agonists acetylcholine (ACh; 10 microM), muscarine (Musc; 20 microM), and oxotremorine methiodide (OxoM; 0.001-20 microM) elicited transient repeatable increases in the intracellular calcium concentration ([Ca(2+)](i)) in approximately 50% of cells. These effects were blocked by the mAChR antagonist atropine methyl nitrate (10 microM). The sources of [Ca(2+)](i) changes included influx from external milieu in 63% of cells and influx from external milieu plus release from internal stores in 27% of cells. The use of specific agonists and antagonists (10 microM M(1) agonist McN-A-343; 10 microM M(2), M(3) antagonists AF-DX 116, 4-DAMP) revealed that M(1), M(2), M(3) subtypes were involved in [Ca(2+)](i) changes. The PLC inhibitor U-73122 (10 microM) abolished OxoM-elicited Ca(2+) responses in the presence of the M(2) antagonist AF-DX 116, suggesting that M(1), M(3), or M(5) mediates [Ca(2+)](i) increases via PLC pathway. ACh (0.1 microM), Musc (10 microM), oxotremorine sesquifumarate (20 microM), and McN-A-343 (1 muM) acting on M(1), M(2), and M(3) mAChR subtypes stimulated ATP release from cultured urothelial cells. In summary, cultured urothelial cells express functional M(1), M(2), and M(3) mAChR subtypes whose activation results in ATP release, possibly through mechanisms involving [Ca(2+)](i) changes.