Stimulated calcium entry and constitutive RhoA kinase activity cause stretch-induced detrusor contraction

Abstract

Urinary bladder wall muscle (i.e., detrusor smooth muscle; DSM) contracts in response to a quick-stretch, but this response is neither fully characterized, nor completely understood at the subcellular level. Strips of rabbit DSM were quick-stretched (5 ms) and held isometric for 10 s to measure the resulting peak quick-stretch contractile response (PQSR). The ability of selective Ca2+ channel blockers and kinase inhibitors to alter the PQSR was measured, and the phosphorylation levels of myosin light chain (MLC) and myosin phosphatase targeting regulatory subunit (MYPT1) were recorded. DSM responded to a quick-stretch with a biphasic response consisting of an initial contraction peaking at 0.24 ± 0.02-fold the maximum KCl-induced contraction (Fo) by 1.48 ± 0.17 s (PQSR) before falling to a weaker tonic (10 s) level (0.12 ± 0.03-fold Fo). The PQSR was dependent on the rate and degree of muscle stretch, displayed a refractory period, and was converted to a sustained response in the presence of muscarinic receptor stimulation. The PQSR was inhibited by nifedipine, 2-aminoethoxydiphenyl borate (2-APB), 100 μM gadolinium and Y-27632, but not by atropine, 10 μM gadolinium, LOE-908, cyclopiazonic acid, or GF-109203X. Y-27632 and nifedipine abolished the increase in MLC phosphorylation induced by a quick-stretch. Y-27632, but not nifedipine, inhibited basal MYPT1 phosphorylation, and a quick-stretch failed to increase phosphorylation of this rhoA kinase (ROCK) substrate above the basal level. These data support the hypothesis that constitutive ROCK activity is required for a quick-stretch to activate Ca2+ entry and cause a myogenic contraction of DSM.

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

“…This would suggest that a constitutively active ROCK is present in bladder smooth muscle. Our results are consistent with previous reports clearly demonstrating the presence of a constitutively active form of ROCK in bladder smooth muscle (9,31,33). Interestingly a constitutively active PKC has also been shown to be present in bladder smooth muscle (33) and vascular smooth muscle (26).…”

“…Our major findings in this report are 1) there are changes in CPI-17 phosphorylation that occur in response to carbachol and there are decreases in basal levels of CPI-17 phosphorylation in the presence of an inhibitor of PKC; 2) there is a dissociation of MLC phosphorylation levels and force by either inhibition of PKC or ROCK; 3) Thr 696 -MYPT1 is not involved in carbachol-induced contractions of bladder smooth muscle; and 4) inhibition of ROCK decreases CPI-17 phosphorylation levels during the tonic phase of a contraction. Our studies also confirm previous reports that inhibition of PKC and ROCK decreases basal and stimulated force production in bladder smooth muscle and that a constitutively active ROCK is present in bladder smooth muscles (9,11,31,32,33).…”

“…It has been shown that both PKC and ROCK are involved in muscarinic receptor-dependent contractions of bladder smooth muscle (11,33). In addition to receptormediated kinases, constitutively active PKC (26,33) and ROCK (9,31) have been shown to be present in vascular and bladder smooth muscles. Therefore, the goal of this study was to test the hypothesis that both CPI-17 phosphorylation and MYPT1 phosphorylation are important in a carbachol-induced contraction of rabbit bladder smooth muscle.…”

Epithelial relaxation mediated by the myosin phosphatase regulator Mypt1 is required for brain ventricle lumen expansion and hindbrain morphogenesis

“…Like non-muscle myosin, smooth muscle is also regulated by Mypt1 and myosin phosphatase (Pfitzer, 2001), and processes akin to epithelial relaxation may exist in vascular smooth muscle (Hirano, 2007), smooth muscle of the urinary bladder (Ding et al, 2009;Poley et al, 2008), gastrointestinal smooth muscle (Huang et al, 2005;Ihara et al, 2007) and in secretion of bile by the gall bladder (CamelloAlmaraz et al, 2009).…”

Inhibitor κB Kinase

“…18,19 These distinct modalities, a feature of certain myosin II motors, 20 may be differently regulated. 19 Moreover, unlike striated muscle that is on or off, constitutive rho kinase activity seems to idle the actomyosin motor of both tonic and phasic smooth muscles, 21,22 although the primary role of rho kinase may be to regulate actin polymerization. 23 Finally, MLCp can involve more than a single phosphorylation site with physiological implications.…”