Cellular regulation of basal tone in internal anal sphincter smooth muscle by RhoA/ROCK

Sustained contractions of smooth muscle cells ( SMC) maintain basal tone in the internal anal sphincter ( IAS). To examine the molecular bases for the myogenic tone in the IAS, the present studies focused on the role of RhoA/ ROCK in the SMC isolated from the IAS vs. the adjoining phasic tissues of the rectal smooth muscle ( RSM) and anococcygeus smooth muscle ( ASM) of rat. We also compared cellular distribution of RhoA/ ROCK, levels of RhoA- GTP, RhoA- Rho guanine nucleotide dissociation inhibitor ( GDI) complex formation, levels of pThr696MYPT1, and SMC relaxation caused by RhoA inhibition. Levels of RhoA/ ROCK were higher at the cell membrane in the IAS SMC compared with those from the RSM and ASM. C3 exoenzyme ( RhoA inhibitor) and Y 27632 ( ROCK inhibitor) caused a concentrationdependent relaxation of the IAS SMC. In addition, active ROCK- II ( primary isoform of ROCK in SMC) caused further shortening in the IAS SMC. C3 exoenzyme increased RhoA- RhoGDI binding and reduced the levels of RhoA- GTP and p(Thr696)-MYPT1. ROCK inhibitor attenuated PKC- induced contractions in IAS SMC. Conversely, a PKC inhibitor ( Go 6850, which causes partial relaxation of the SMC) had no significant effect on ROCK-II-induced contractions. Further experiments showed the highest levels of RhoA, active form of RhoA ( RhoA- GTP), ROCK- II, 20-kDa myosin regulatory light chain ( MLC20), phospho- MYPT1, and phospho- MLC20 in the IAS vs. RSM and ASM SMC. However, the trend was the reverse with the levels of inactive RhoA ( GDP- RhoA- RhoGDI complex) and MYPT1. We conclude that RhoA/ ROCK play a critical role in maintenance of spontaneous tone in the IAS SMC via inhibition of myosin light chain phosphatase.