The urothelium serves as an active barrier to prevent pathogens and chemical stimuli within the urine from influencing urinary bladder function. However, certain pathogens can activate inflammatory signaling pathways that lead to urothelial cell damage, dysregulation of adenosine triphosphate (ATP) release, and neural activation. This is evident in urinary tract infections, interstitial cystitis, pelvic pain, and painful bladder syndrome. Current animal models used to induce interstitial cystitis or bacterial infection implicate mast cell degranulation as a leading cause of hypersensitivity that induces lower urinary tract symptoms. Thus, we hypothesized that phasic contractions caused by the mast cell activator compound 48/80 were due to mast cell degranulation and the subsequent release of prostaglandin E2 from the urothelium. Based on this hypothesis, we also examined the effects of compound 48/80 on urinary bladder smooth muscle (UBSM) strips from non-diseased mast cell deficient and C57BL/6 mice to establish a physiological mechanism by which mast degranulation can cause smooth muscle excitability.

Based on our previous findings, we focused our efforts on both mast cells and the urothelium to determine the site of release of inflammatory mediators. We utilized pharmacological inhibitors and antagonists to determine the signaling molecule responsible for compound 48/80-induced contractions. The selection of each inhibitor and antagonist was based on known signaling pathways that are responsible for inducing inflammation-driven contractility. In the urinary bladder, purinergic activation via the release of ATP causes the synthesis of prostaglandins resulting in UBSM contraction. Therefore, isometric contractility was performed with urothelium denuded and intact urinary bladder smooth muscle (UBSM) strips from C57Bl/6 and mast cell deficient c-KitW-sh/W-sh (Sash) mice. UBSM strips from C57Bl/6 male mice (11 – 18 weeks old) were incubated with vehicle or the following drugs prior to exposure to compound 48/80: the nonsteroidal anti-inflammatory and cyclooxygenase (COX) inhibitor indomethacin (5 µM); the prostaglandin EP1 receptor antagonist SC51089 (10 µM); the non-selective P2 purinergic antagonist PPADS (100 µM); and the Gq/11 signaling inhibitor YM254890 (1 µM). Two urothelium-intact strip and two urothelium-denuded strips were isolated from each mouse bladder, and experiments were performed in parallel. These experiments were then repeated under the same conditions in male Sash mice (11 weeks old).

In UBSM strips from C57Bl/6 mice, compound 48/80 caused both an increase in basal UBSM tone as well as an increase in phasic contractions. Removal of the urothelium significantly reduced the amplitude and occurrence of phasic contractions (p=0.005 vs urothelium-intact). In UBSM strips from Sash mice, compound 48/80 still caused both an increase in basal UBSM tone as well as an increase in phasic contractions, which were unchanged vs C57Bl/6 mice. Removal of the urothelium also significantly reduced the amplitude and occurrence of phasic contractions in Sash mice (p=0.004 vs urothelium-intact). SC51089 had no effect on phasic contractions induced by compound 48/80 (p=0.420). However, indomethacin (p=0.04), PPADS (p=0.01), and YM254890 (p=0.028) all significantly reduced basal tone and the amplitude and occurrence of phasic contractions.

Our data suggest that the basic secretagogue compound 48/80 is increasing phasic contractions via activation of urothelial cell signaling that is causing the release of ATP and activation of a cyclooxygenase-mediated pathway to increase synthesis of pro-contractile mediators. In addition, compound 48/80-induced contractions are dependent on Gq signaling, which could suggest a synergistic pathway. As compound 48/80 is known to activate G protein-coupled receptors to activate mast cells, our data further suggest the targets of compound 48/80 are not specific to mast cells. Although the exact signaling molecule and pathway responsible remains unclear, our data support a feed-forward mechanism that is increasing spontaneous mechanical behavior.

We have discovered that compound 48/80 induced UBSM contractions are independent of mast degranulation but depend on the urothelium. These findings show that basic secretagogues, such as compound 48/80, may not require mast cells to markedly alter urinary bladder function in response to an inflammatory insult. Our results also shed new light on the crosstalk between UBSM and urothelium and give further insight on potential therapeutic targets for inflammatory bladder dysfunction.