Cystitis-Related Bladder Pain Involves ATP-Dependent HMGB1 Release from Macrophages and Its Downstream H2S/Cav3.2 Signaling in Mice

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Ca(v)3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-gamma-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Ca(v)3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Ca(v)3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X(4)/P2X(7) receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X(7)/P2X(4), and caused HMGB1 release via P2X(7)in RAW264.7 cells, which was dependent on p38MAPK/NF-kappa B signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Ca(v)3.2-dependent nociceptor excitation, resulting in bladder pain.