Soluble Guanylyl Cyclase (sGC) Degradation and Impairment of Nitric Oxide-Mediated Responses in Urethra from Obese Mice: Reversal by the sGC Activator BAY 60-2770

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined the urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study aimed to investigate the USM relaxant machinery in obese mice, and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 rescues the urethral reactivity through improvement of sGC-cGMP signaling. Male C57BL/6 mice were fed for 12 weeks with high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg/day, 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS) and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO-donors (S-nitrosoglutathione and glyceryl trinitrate) and BAY 41-2272 (sGC stimulator) were markedly reduced in obese compared with lean mice. As opposed, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice ( P <0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ (10 μ M) potentiated BAY 60-2770-induced USM responses in lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, whereas protein expression of β 1 sGC subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and downregulation of sGC-cGMP signalling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice. One concentration–response curve to each relaxing agent was obtained in each urethral preparation. For BAY 41-2272 and BAY 60-2770, control experiments were carried out in urethral rings in the presence of 0.22 - 0.32% DMSO (vehicle used to dissolve these drugs). In separate urethral preparations, concentration-response curves were repeated in the presence of the sGC inhibitor ODQ (10 pre-incubated for 30 min. Nonlinear regression analysis to determine the pEC 50 was carried out using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA) with the constraint that F = 0. All concentration–response data were evaluated for a ﬁt to a logistics function in the form: E = Emax/([1 + (10c/10x)n] + F), where E is the maximum response produced by agonists; c is the logarithm of the EC 50 , the concentration of drug that produces a half-maximal response; x is the logarithm of the concentration of the drug; the exponential term, n, is a curve-ﬁtting parameter that deﬁnes the slope of the concentration–response line, and F is the response observed in the absence of added drug. Relaxing responses were calculated as percentages of the maximal changes from the steady-state contraction produced by phenylephrine (10 M) in each tissue. Data are shown as the percentage of relaxation of n experiments, expressed as the mean ± S.E. M. EC 50 values are presented as the negative logarithm (pEC50) and calculated by a fitting concentration–response relationship to a sigmoidal model of the form log-concentrations vs response using GraphPad Software.