Poster 8: The Influence of Ovariectomy on the Rhoa/Rho-Kinase Pathway of Smooth Muscle Calcium Sensitization in the Female Rat Bladder Dome

Abstract

OBJECTIVES: Detrusor muscle overactivity is detected in 23% to 42% of incontinent women, the highest incidence reported with increasing age. Despite this wide prevalence, the actual molecular pathogenesis leading to overactive bladder is largely unknown. We propose that alterations in smooth muscle (SM) proteins may play a role. The goal of our study was to examine the expression of the 2 smooth muscle calcium-sensitizing enzymes, Rho-kinase alpha and beta (ROKα and β), and their upstream activator, RhoA, in the bladder dome of ovariectomized female rats versus sham-operated controls. METHODS: Twelve female Sprague-Dawley rats weighing 275–300 g were stratified into one of 2 groups, 3-week ovariectomized (group I) and 3-week sham control (group II). After acclimation, both groups underwent a ventral midline incision under anesthesia. Group I also underwent bilateral ovariectomy. Three weeks after surgery, the animals were killed by CO2 asphyxia and the dome of the bladder was removed, its epithelium and serosa were dissected, and the remaining tissue was flash-frozen in liquid nitrogen. Total RNA was isolated, the RNA transcribed into cDNA, and real-time polymerase chain reaction was performed using primer pairs specific for RhoA, ROKα, and ROKβ using the ABI 7300 thermocycler, with α-actin amplified as an internal control. RESULTS: There was no statistical significance in the mean age or weight at killing between the 2 groups. However, our analysis revealed a 3.2-cycle decrease (corresponding to a roughly 8-fold increase) in RhoA expression and a 4.2-cycle decrease (a roughly 16-fold increase) in ROKα expression in the ovariectomized specimens compared with shams. There was no statistically significant change in ROKβ expression in response to ovariectomy. CONCLUSIONS: Ovariectomy induced an upregulation in the expression of the Rho-kinase pathway (in particular ROKα) in the bladder dome of female rats. An increase in the RhoA/Rho-kinase pathway, which sensitizes smooth muscle to calcium, would be expected to increase the tone of the bladder detrusor SM in the ovariectomized rats, suggesting a possible involvement of this pathway in the molecular mechanism of bladder overactivity in postmenopausal women.