PROJECT SUMMARY/ABSTRACT
The bladder must refrain from premature contraction during filling and empty when critical intravesical volume
and pressure are reached. Abnormal continence or voiding of urine are frequently associated with abnormal
excitability of the detrusor smooth muscle (DSM) in the course of bladder filling. Current therapies are largely
ineffective and frequently have intolerable side effects. There is a pressing need to better understand the
fundamental mechanisms of regulation of bladder function during filling that may yield novel ideas for more
efficient control of bladder excitability. Excitatory purines that increase DSM contractility (e.g., ATP and ADP)
and inhibitory purines that decrease DSM contractility (e.g., NAD, ADP-ribose, AMP and adenosine) are released
from the urothelium and form “a regulatory purine pool” deep in the bladder wall. The relative composition of this
pool (e.g., inhibitory vs. excitatory) might be changing during bladder filling to enable adequate DSM excitability.
However, purine-mediated local mechanisms of signaling between the urothelium and DSM during filling are not
understood. This project will investigate 1) several mechanisms - release, metabolism and transurothelial
transport - that determine the type and relative amount of purine mediators available in suburothelium
(SubU)/lamina propria (LP) during filling and 2) influences of extracellular purines on non-neural types of cells in
the bladder wall that regulate DSM excitability. Specific Aim 1 will test the hypothesis that asymmetrical
availability of purines leads to a higher ratio of inhibitory/excitatory purines in SubU/LP during the storage phase
of bladder filling whereas reduction of this ratio at high volume and pressure facilitates micturition. ATP, ADP,
NAD, ADP-ribose, AMP and adenosine will be examined simultaneously in SubU/LP and in lumen during filling.
Specific Aim 2 will test the hypothesis that metabolism and transurothelial transport of purines regulate adequate
purine availability in the SubU/LP during bladder filling. Specific Aim 3 will test the hypothesis that urothelial
purines contribute to the intrinsic control of bladder excitability during filling by affecting urothelial cells,
submucosal PDGFR¿+ cells and DSM cells. To obtain direct access to SubU/LP, we will use a decentralized (ex
vivo) bladder model with DSM removed and we will perform in vivo and ex vivo microdialysis of the bladder wall.
We will use analytical chemistry, electrophysiology, molecular biology, protein biochemistry, and functional and
Ca2+ imaging methodologies, including expression of optogenetic sensors in selected cell types in the bladder
wall. Studies will employ transgenic mice such as Pdgfr¿egfp/+, smMHC-GCaMP6f, PDGFR¿-GCaMP6,
Trpv4eGFP, AQP3-GCaMP6m mice and mice with specific gene deletions. Key mechanisms will be validated in
bladders from Cynomolgus monkeys (Macaca fascicularis) to determine how knowledge obtained in mouse
bladder translates to the primate bladder. At the end of the project period, we will understand the biological
significance of urothelial purinergic signaling for mechanosensitive connectivity between the urothelium and DSM
and we may identify novel mechanistic targets for the treatment of anomalous bladder excitability.