Effects of Rho‐kinase inhibition on myosin light chain phosphorylation and obstruction‐induced detrusor overactivity

Abstract

Abbreviations & Acronyms DMSO = dimethylsulfoxide DO = detrusor overactivity DSM = detrusor smooth muscle EC50 = half maximal effective concentration GSK = GlaxoSmithKlein IEF = isoelectric focusing OAB = overactive bladder MLC20 = myosin light chain 20 kDa MLCP = myosin light chain phosphatase MYPT1 = myosin phosphatase 1 pBOO = partial bladder outlet obstruction ROK = Rho-kinase SHR = spontaneously hypertensive rats Objectives: To study the relationship between myosin light chain phosphorylation of the detrusor muscle and spontaneous smooth muscle contractions in a rabbit model of partial outlet obstruction. Methods: New Zealand white rabbit urinary bladders were partially obstructed for 2 weeks. Rabbits were euthanized, detrusor muscle strips were hung on a force transducer and spontaneous activity was measured at varying concentrations (0-0.03 μM/L) of the Rho-kinase inhibitors GSK 576371 or 0.01 μM/L Y27632. Basal myosin light chain phosphorylation was measured by 2-D gel electrophoresis in control and GSK 576371-treated strips. Results: Both drugs suppressed the force of spontaneous contractions, whereas GSK 576371 had a more profound effect on the frequency of the contractions. The IC50 values for the inhibition of frequency and force of spontaneous contractions were 0.17 μM/L and 0.023 μM/L for GSK 576371, respectively. The compound significantly decreased the basal myosin light chain phosphorylation from 28.0 ± 3.9% to 13.5 ± 1.9% (P < 0.05). At 0.01 μM/L, GSK 576371 inhibited spontaneous bladder overactivity by 50%, but inhibited carbacholelicited contractions force by just 25%. Conclusions: These data suggest that Rho-kinase regulation of myosin light chain phosphorylation contributes to the spontaneous detrusor activity induced by obstruction. This finding could have therapeutic implications by providing another therapeutic option for myogenic, overactive bladder.

Benefits and limitations of animal models in partial bladder outlet obstruction for translational research

“…Elevations in reactive oxygen species and reactive nitrogen species, and changes in the activity of anti‐oxidant mechanisms are induced by pBOO models . Rho‐kinase inhibitors significantly suppressed the force of the spontaneous contractions …”

Protein kinase C modulates frequency of micturition and non-voiding contractions in the urinary bladder via neuronal and myogenic mechanisms

“…Among identified mechanisms are neurogenic and myogenic alterations of detrusor contractility, as well as changes in bladder stiffness, and ion channels associated with partial bladder outlet obstruction (PBOO), and other pathological conditions of the urinary bladder [ 3 , 8 - 15 ]. It was previously established that a number of DSM pathologies are linked to the changes in signaling proteins including, but not limited to, protein kinase C (PKC), Rho-associated kinase (ROK), and large conductance Ca 2+ -activated potassium (BK) channels [ 5 , 13 , 16 - 26 ]. Alterations in smooth muscle myosin isoforms from the fast phasic isoform to a more tonic one in PBOO [ 27 , 28 ] is correlated with aberrant calcium signaling [ 29 ], and higher resting levels of myosin light chain phosphorylation (MLCP) associated with bladder overactivity [ 13 ].…”

“…It was previously established that a number of DSM pathologies are linked to the changes in signaling proteins including, but not limited to, protein kinase C (PKC), Rho-associated kinase (ROK), and large conductance Ca 2+ -activated potassium (BK) channels [ 5 , 13 , 16 - 26 ]. Alterations in smooth muscle myosin isoforms from the fast phasic isoform to a more tonic one in PBOO [ 27 , 28 ] is correlated with aberrant calcium signaling [ 29 ], and higher resting levels of myosin light chain phosphorylation (MLCP) associated with bladder overactivity [ 13 ]. It has also been suggested that overactive bladder associated with hypertrophic changes in the smooth muscle may be due to a switch from M 3 to M 2 muscarinic receptor signaling which tends to show increased sensitivity [ 30 - 32 ].…”

Does altered myogenic activity contribute to OAB symptoms from detrusor overactivity? ICI-RS 2013

“…ROCK over‐expression suggests that ROCK inhibitors, such as Y27632, may potentially be a treatment option for DO, and they have been proposed as a therapy in other disorders associated with smooth muscle dysfunction such as hypertension and atherogenesis and GI tract disorders including: recto‐anal incontinence; Hirschsprung's disease (HPD), and gastro‐oesophageal reflux disease . Recently, the ROCK inhibitors Y27632 and GSK‐576371 have been shown to suppress spontaneous bladder overactivity using a rat model of outflow tract obstruction …”