[Background and aims] Bladder pain accompanying cyclophosphamide (CPA)-evoked cystitis in mice involves the upregulation of cystathionine-γ-lyase (CSE), an H2S-forming enzyme, and subsequent increase in the activity and expression of Cav3.2 T-type Ca2+ channels, known as a target for H2S (Br J Pharmacol 167, 917, 2012; Toxicology 393, 102, 2018). We have also shown the involvement of RAGE activation by high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, in the cystitis-related bladder pain (Neuropharmacology 79, 112, 2014). The present study thus examined the relationship between HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in bladder pain signaling. [Methods] Female mice received i.p. CPA at 400 mg/kg, and bladder pain (nociceptive behavior and referred hyperalgesia) was assessed 3.5-4 h later. Protein levels of CSE were determined by Western blotting or immunohistochemistry. Macrophages (Mφ) accumulating in the bladder tissue were detected by immunohistochemistry. [Results] The cystitis-related bladder pain caused by CPA in mice was inhibited by (2R/S)-6-prenylnaringenin and KTt-45, novel T-channel blockers, or by genetic deletion of Cav3.2. The CPA-evoked bladder pain and upregulation of CSE in the bladder tissue were abolished by an anti-HMGB1 neutralizing antibody, FPS-ZM1, a RAGE antagonist, ethyl pyruvate, an inhibitor of HMGB1 release from Mφ or liposomal clodronate, a Mφ depletor. Mφ accumulation was detected in the bladder mucosal layer after CPA treatment. [Conclusions] Our data suggest that Mφ-derived HMGB1 following CPA treatment triggers RAGE-dependent CSE upregulation in the bladder, and the increased H2S generated by CSE enhances Cav3.2 channel activity, which in turn induces nociceptor excitation followed by bladder pain.

CROSSTALK BETWEEN THE HMGB1/RAGE AND CSE/H2S/CAV3.2 PATHWAYS INVOLVED IN CYSTITIS-RELATED BLADDER PAIN IN MICE