Bladder inflammation is a major feature of urological disorders such as Overactive Bladder (OAB) and Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS), which affect millions of people and lack consistently effective treatments. Previous studies have identified purinergic signaling as a key contributor to bladder pathology, while research from our laboratory has shown that ATP release from the urothelium via lysosomal exocytosis promotes bladder inflammation and overactivity. This ATP release is regulated by lysosomal calcium (Ca²⁺) signaling through TRPML1 channels and is often triggered by intracellular oxidative stress, although the signaling pathways connecting oxidative stress to lysosomal Ca²⁺ release remain unclear. This project investigates whether oxidative stress induced by biologically active molecules (Menadione, Angiotensin II, ML-SA1, and ML-SA5) activates TRPML1-mediated lysosomal Ca²⁺ release in urothelial cells. Using Fura-2 calcium imaging, we measured intracellular Ca²⁺ responses and evaluated the effects of the TRPML1 inhibitor ML-SI3 to determine channel involvement. We also examined the roles of BK potassium channels and the CFTR chloride channel in regulating lysosomal ATP release. By clarifying how oxidative stress controls lysosomal Ca²⁺ signaling and ATP release, this research advances our understanding of bladder inflammation and may identify novel therapeutic targets for OAB, IC/BPS, and related bladder disorders.
