Changes in Adenosine Triphosphate-stimulated ATP Release Suggest Association Between Cytokine and Purinergic Signaling in Bladder Urothelial Cells

Objectives: To determine whether antiproliferative factor (APF) or epidermal growth factor (EGF) can induce changes in purinergic signaling in normal bladder urothelial cells (BUCs) and/or whether antagonizing EGF activity or blocking adenosine triphosphate (ATP)-purinergic receptors can induce changes in purinergic signaling in interstitial cystitis (IC) cells. Methods: IC and normal BUCs were obtained from patients' bladder biopsy specimens. IC BUCs were treated with genistein, which antagonizes EGF's activity, and normal BUCs were treated with EGF, mock APF, or APF. Suramin, which antagonizes ATP activity, was used to treat the APF-treated normal BUCs. ATP release was determined by stimulating the BUCs with 30 μM ATP and then collecting the supernatant for a 3-hour period. ATP quantification was measured by luciferin-luciferase assay. Purinergic receptor P2X, ligand-gated ion channel, 3 (P2X3) expression on BUCs was determined by fluorescence-activated cell sorting. Results: Genistein treatment of IC BUCs resulted in significantly decreased ATP release, thus reverting IC cells to a normal purinergic signaling phenotype. Conversely, normal BUCs treated with EGF or APF resulted in significantly increased ATP release and P2X3 expression, converting normal BUCs to an IC phenotype. Also, suramin treatment of APF-treated normal BUCs significantly reduced ATP release. Conclusions: Genistein and suramin reversed the augmented ATP release in IC BUCs and APF-treated normal BUCs, respectively, suggesting the possibility of intravesical use of these agents in IC treatment. EGF and APF induced augmented purinergic signaling in normal BUCs, as determined by increased ATP release and increased P2X3 expression. These data suggest an association between cytokines and purinergic signaling in human BUCs that should be explored further.