Submitted on March 22, 2006
Accepted on April 27, 2006
Spontaneously Tonic Smooth Muscle has Characteristically Higher Levels of RhoA/ROK as Compared with the Phasic Smooth Muscle
Chirag A Patel1 and Satish Rattan1*
1 Medicine/Division of Gastroenterology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
* To whom correspondence should be addressed. E-mail: satish.rattan{at}jefferson.edu.
The internal anal sphincter (IAS) tone is important for the rectoanal continence. RhoA/Rho kinase (ROK) pathway have been associated with the agonist-induced sustained contraction of the smooth muscle (SM), but its role in the spontaneously tonic smooth muscle is not known. Present studies compared expression of different components of RhoA/ROK pathway between the IAS (a truly tonic SM), the RSM (a mixture of phasic and tonic), and anococcygeus (ASM) (a purely phasic SM) of rat. RT-PCR and western blots analyses were performed to determine RhoA, ROCK-II, CPI-17, MYPT1, myosin light chain 20 (MLC20). Phosphorylated- CPI-17 at Threonine-38 residue (pThr38-CPI-17), MYPT1 at Threonine-696 residue (pThr696-MYPT1) and MLC20 at Threonine-18/Serine-19 residues (pThr18/Ser19-MLC20) were also determined in the basal state and after pretreatment with the ROK inhibitor Y 27632. In addition, we compared the effect of Y 27632, on the basal isometric tension and ROK activity in the IAS vs. the RSM. Our data show highest levels of RhoA, ROCK-II, CPI-17, MLC20, and of phospho- MYPT1, CPI-17 and MLC20 in the IAS followed by in the RSM and ASM. Conversely, MYPT1 levels were lowest in the IAS and highest in the ASM. In the IAS, Y 27632 caused a concentration-dependent decrease in the basal tone and in the levels of phospho- MYPT1, CPI-17, and MLC20, and ROK activity. We conclude that RhoA/ROK play a critical role in the basal tone in the IAS by the inhibition of myosin light chain phosphatase (MLCP) via the phosphorylation of MYPT1 and CPI-17.
