A balance between stiffness and compliance is essential to normal bladder function. Inflammatory processes, such as mast cell activation, can affect the integrity of the bladder wall and shift this balance toward fibrosis and increased stiffness. The resultant bladder wall remodeling ultimately leads to many lower urinary tract symptoms. Compound 48/80 is a basic secretagogue that rapidly degranulates mast cells and brings about release of multiple inflammatory mediators. These mediators, including leukotrienes, prostaglandins, and matrix metalloproteases (MMPs), are implicated in many downstream pathways that alter urinary bladder smooth muscle contractility as well as wall compliance. In this study, we explored if and how compound 48/80 alters urinary bladder smooth muscle contractility and wall compliance in order to identify pro-inflammatory mediators that may be released due to the action of compound 48/80.

Whole mouse bladders were dissected and mounted in the custom designed Pentaplanar Reflected Image Macroscopy (PRIM) System for simultaneous measurement of transient pressure events, bladder wall deformation, bladder volume and intravesical pressure during ex vivo bladder filling. Bladders were filled at a constant rate (30 µl/min) until intravesical pressure reached 25 mmHg, at which point bladders were emptied and allowed to recover for approximately 10 minutes. Pressure and video images were recorded simultaneously at a rate of 10 Hz. Fill/empty cycles (approximately 20 minutes) were then repeated in the presence or absence of antagonist prior to the addition of vehicle or the mast cell activator compound 48/80 (10 µg/ml). Antagonists used were: broad spectrum MMP inhibitor doxycycline (20 µM); leukotriene receptor antagonist montelukast (10 µM); and cyclooxeganse inhibitor indomethacin (10 µM). Using the recorded intravesical pressures, infusion volumes, image data, we next calculated the bladder wall compliance (defined as the stress versus stretch relationship of the bladder wall).

During filling, baseline intravesical pressure remained low and increased slowly over a relatively large infused volume (~350 µl), after which intravesical pressure began to increase rapidly. Transient contractions began on the lateral sides of the bladder wall near the trigone, expanded upwards as the bladder filled, and corresponded to transient changes in intravesical pressure (0-2 mmHg). As with pressure, wall stress was relatively low over a large change in stretch during filling. Compound 48/80 alone significantly increased the amplitude and leading slope of transient contractions while increasing bladder wall compliance. The broad spectrum MMP inhibitor doxycycline prevented the increase in compliance caused by compound 48/80; however, the amplitude and leading slope of transient contractions was unaffected. The leukotriene receptor antagonist montelukast did not inhibit any effects of compound 48/80. Interestingly, the non-selective cyclooxygenase inhibitor indomethacin prevented all effects of compound 48/80.

The stress and stretch curves show that wall compliance is non-linear: compliance is high until larger infused volumes, after which bladder wall compliance rapidly decreases. The results of the study imply that the mechanism through which compound 48/80 acts is two-fold and parallel. Compound 48/80 is first stimulating the release/synthesis of prostaglandins, which then cause the activation of matrix metalloproteases that rapidly degrade collagen in the extracellular matrix to increase compliance. Further studies can be done with more specific MMP inhibitors, for example marimastat, to confirm the dependence on individual MMPs. Prostaglandins are simultaneously increasing the amplitude and leading slope of transient contractions. The change in urinary bladder smooth muscle contractility is not a product of the change in compliance, since inhibiting MMPs did not affect the increased transient pressure event amplitude or leading slope caused by compound 48/80. For the next series of experiments, qRT-PCR will be performed in the urinary bladder smooth muscle and urothelial layers to compare COX isoform expression and identify the specific COX enzyme that is involved in the mechanism.

Our study discovered that the effects of Compound 48/80 on the urinary bladder are multi-faceted, in that it rapidly activates mast cells to release prostaglandins, which then increase bladder smooth muscle contractility and activate MMP-mediated increases in bladder wall compliance. Thus, bladders can show signs of overactivity while actually increasing in compliance. Furthermore, changes in compliance and urinary bladder contractility can occur simultaneously and independently. This pathway paves the way for reframing our knowledge on treatments offered for bladder dysfunction that is accompanied by inflammation.