Spontaneously tonic smooth muscle has characteristically higher levels of RhoA/ROK compared with the phasic smooth muscle.
The internal anal sphincter (IAS) tone is important for the rectoanal continence. The RhoA/Rho kinase (ROK) pathway has been associated with the agonist-induced sustained contraction of the smooth muscle, but its role in the spontaneously tonic smooth muscle is not known. Present studies compared expression of different components of the RhoA/ROK pathway between the IAS (a truly tonic SM), the rectal smooth muscle (RSM) (a mixture of phasic and tonic), and anococcygeus smooth muscle (ASM) (a purely phasic SM) of rat. RT-PCR and Western blot analyses were performed to determine RhoA, ROCK-II, CPI-17, MYPT1, and myosin light-chain 20 (MLC20). Phosphorylated CPI-17 at threonine-38 residue (p(Thr38)-CPI-17), MYPT1 at threonine-696 residue (p(Thr696)-MYPT1), and MLC20 at threonine-18/serine-19 residues (p(Thr18/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 the 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, levels of phospho-MYPT1, -CPI-17, and -MLC20, and ROK activity. We conclude that RhoA/ROK plays a critical role in the basal tone in the IAS by the inhibition of MLC phosphatase via the phosphorylation of MYPT1 and CPI-17.
Patel
Chirag A
CA
Department of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson University, 1025 Walnut St., Rm. 901, Philadelphia, PA 19107, USA.
Rattan
Satish
S
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D007537:Isometric Contraction / Q000502:physiology; D008297:Male; D009119:Muscle Contraction / Q000502:physiology; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics; D009129:Muscle Tonus / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000235:genetics; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054460:rho-Associated Kinases
D000577:Amides; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D011725:Pyridines; C108830:Y 27632; D012313:RNA; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1