Adjustable passive stiffness in mouse bladder: regulated by Rho kinase and elevated following partial bladder outlet obstruction

Abstract

Detrusor smooth muscle (DSM) contributes to bladder wall tension during filling, and bladder wall deformation affects the signaling system that leads to urgency. The length-passive tension (L-T(p)) relationship in rabbit DSM can adapt with length changes over time and exhibits adjustable passive stiffness (APS) characterized by a L-T(p) curve that is a function of both activation and strain history. Muscle activation with KCl, carbachol (CCh), or prostaglandin E(2) at short muscle lengths can increase APS that is revealed by elevated pseudo-steady-state T(p) at longer lengths compared with prior T(p) measurements at those lengths, and APS generation is inhibited by the Rho Kinase (ROCK) inhibitor H-1152. In the current study, mouse bladder strips exhibited both KCl- and CCh-induced APS. Whole mouse bladders demonstrated APS which was measured as an increase in pressure during passive filling in calcium-free solution following CCh precontraction compared with pressure during filling without precontraction. In addition, CCh-induced APS in whole mouse bladder was inhibited by H-1152, indicating that ROCK activity may regulate bladder compliance during filling. Furthermore, APS in whole mouse bladder was elevated 2 wk after partial bladder outlet obstruction, suggesting that APS may be relevant in diseases affecting bladder mechanics. The presence of APS in mouse bladder will permit future studies of APS regulatory pathways and potential alterations of APS in disease models using knockout transgenetic mice.

Fig. 1.

A: loading/unloading protocol for measuring passive tension (T_p) due to KCl-induced adjustable passive stiffness (APS) in mouse bladder strips. B: typical T_p tracings. C: length-passive tension (L-T_p) loading and unloading curves in Ca²⁺-free solution (0-Ca) following incubation in 0-Ca or modified physiological salt solution (PSS) at 50% maximum length (L_max) or following precontraction with KCl at 50% L_max [T_p values normalized to reference tension (T_ref), n = 4]. D−F: T_p at 80, 90, and 95% L_max normalized to T_p during loading at that length following KCl precontraction (T_p/T_{p_load_KCl}; means ± SE, * indicates value significantly less than 1.0, and Ω indicates unloading value significantly less than the corresponding loading value, P < 0.025, n = 4).

Fig. 2.

A: loading/unloading protocol for measuring T_p due to carbachol (CCh)-induced APS in mouse bladder strips. B: L-T_p loading and unloading curves following 10-μM CCh precontractions (CCh_A and CCh_B) or no precontractions (incubation in 0-Ca, 0-Ca_A, and 0-Ca_B) at 50% L_max (means ± SE, T_p values normalized to T_ref, n = 3). C–E: T_p at 70, 80, and 90% L_max normalized to T_p at that length following CCh precontraction (T_p/T_{p_load_CChA}; * indicates value significantly less than 1.0, and Ω indicates an unloading value significantly less than the corresponding loading value, P < 0.025, n = 3).

Fig. 3.

Passive and active pressure-volume curves for female mouse bladders (means ± SE for pressure and volume, * indicates passive pressure value significantly greater than passive pressure at 20 μl, and ψ indicates that active pressure at 72.5 μl was significantly less than at 59 μl, paired t-test, P < 0.05, n = 10).

Fig. 4.

A: passive filling pressure in 0-Ca during 10-min fills with (stiff) and without (compliant) a 1-μM CCh precontraction at 10% V_ref. The pressure difference (P_APS = stiff - compliant) was attributed to APS. B: passive filling pressure during 10- and 55-min fills with and without a 1-μM CCh precontraction.

Fig. 5.

Passive filling pressure data at 40% reference volume (V_ref) for 3 groups of bladders: control, staurosporine (STP), and H-1152. Each group underwent 4 passive filling cycles from 10% to 100% V_ref, with the 1st and 3rd following precontraction with 10 μM CCh and the 2nd and 4th following incubation in 0-Ca with no precontraction (CCh_A, 0-Ca_A, CCh_B, and 0-Ca_B). A: pressure in control bladders at 40% V_ref during each of the 4 filling cycles (means ± SE, * indicates 0-Ca value significantly less than the prior CCh value, P < 0.05, n = 6). B: effects of 10 μM STP and 1.0 μM H-1152 during CCh stimulation on the subsequent normalized passive pressure at 40% V_ref during the 3rd fill-empty cycle (CCh_B ± drug; * indicates values significantly less than 1.0, P < 0.05, n = 6 control, n = 3 STP and H1152). C: effects of 10 μM STP and 1.0 μM H-1152 during CCh stimulation before the 3rd cycle on normalized passive pressure attributed to APS (P_APS) at 40% V_ref during the 3rd passive filling cycle (P_{APS_A} = CCh_A − 0-Ca_A, P_{APS_B} ± drug = CCh_B ± drug − 0-Ca_B, Ω and * indicate values significantly greater than or less than 1.0, respectively, P < 0.05, n = 6 control, n = 3 STP and H-1152).

Fig. 6.

A: peak pressure attributed to APS during 4 passive bladder filling cycles from 10% to 100% V_ref, with the 1st and 3rd following precontraction with 10 μM CCh and the 2nd and 4th following incubation in 0-Ca with no precontraction (CCh_A, 0-Ca_A, CCh_B, and 0-Ca_B), with P_{APS_A} = CCh_A − 0-Ca_A, and P_{APS_B} = CCh_B − 0-Ca_B (means ± SE, Ψ indicates significant difference, P < 0.025, n = 6). B: peak P_APS as a fraction of P_ref (Ψ indicates significant difference, P < 0.025, n = 6). C: effect of 10 μM STP or 1.0 μM H-1152 during CCh stimulation before the 3rd fill-empty cycle (CCh_B ± drug) on the subsequent normalized peak P_APS during passive bladder filling (P_{APS_B} ± drug = CCh_B ± drug − 0-Ca_B. Ω and * indicate values significantly greater than or less than 1.0, respectively, P < 0.05, n = 6 control, n = 3 STP and H-1152).

Fig. 7.

Passive filling pressure in deionized H₂O at 40% V_ref (H₂O_A and H₂O_B) compared with filling pressure in 0-Ca with (CCh_A and CCh_B) and without (0-Ca_A and 0-Ca_B) 10 μM CCh precontraction at 10% V_ref (means ± SE, values normalized to CCh_A, * indicates value significantly less than 1.0, and Ω indicates value significantly less than the 0-Ca_B value, n = 3, P < 0.025).

Fig. 8.

A: passive and active pressure-volume curves for male control (means ± SE, n = 7) and partial bladder outlet obstruction (PBOO) mouse bladders. PBOO data were divided into distinct 2 groups: those categorized as compensated with V_ref < 100 μl (compensated PBOO, n = 4), and those categorized as decompensated with V_ref > 165 μl (decompensated PBOO, n = 2). Passive pressure for the compensated PBOO group was significantly greater than the control group at all 10-μl volume increments ≥ 20 μl (P < 0.05). Active pressure for the compensated PBOO group was less than the control group and this difference approached statistical significance at volumes ≥ 80 μl (P < 0.059). B–E: V_ref, P_ref, wet weight, and P_ref/weight, respectively. * Indicates a compensated PBOO value significantly less than the control value, P < 0.05.

Fig. 9.

A–B: peak P_APS and peak P_APS/(P_ref/wt) during passive filling following precontraction with KCl or 1 mM CCh for control, compensated PBOO, and decompensated PBOO mouse bladders (means ± SE, n = 7, 4, and 2, respectively). * Indicates a compensated PBOO value significantly greater than the control value, P < 0.05.