title: RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter.
abstract: The knowledge of molecular control mechanisms underlying the basal tone in the intact human internal anal sphincter (IAS) is critical for the pathophysiology and rational therapy for a number of debilitating rectoanal motility disorders. We determined the role of RhoA/ROCK and PKC pathways by comparing the effects of ROCK- and PKC-selective inhibitors Y 27632 and Gö 6850 (10(-8) to 10(-4) M), respectively, on the basal tone in the IAS vs. the rectal smooth muscle (RSM). Western blot studies were performed to determine the levels of RhoA/ROCK II, PKC-α, MYPT1, CPI-17, and MLC(20) in the unphosphorylated and phosphorylated forms, in the IAS vs. RSM. Confocal microscopic studies validated the membrane distribution of ROCK II. Finally, to confirm a direct relationship, we examined the enzymatic activities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC(20), before and after Y 27632 and Gö 6850. Data show higher levels of RhoA/ROCK II and related downstream signal transduction proteins in the IAS vs. RSM. In addition, data show a significant correlation between the active RhoA/ROCK levels, ROCK enzymatic activity, downstream proteins, and basal IAS tone, before and after ROCK inhibitor. From these data we conclude 1) RhoA/ROCK and downstream signaling are constitutively active in the IAS, and this pathway (in contrast with PKC) is the critical determinant of the basal tone in intact human IAS; and 2) RhoA and ROCK are potential therapeutic targets for a number of rectoanal motility disorders for which currently there is no satisfactory treatment.
authors.affiliation: Dept. of Medicine, Division of Gastroenterology & Hepatology, Philadelphia, PA 19107, USA. satish.rattan@jefferson.edu
authors.forename: Satish
authors.initials: S
authors.lastname: Rattan
authors.forename: Jagmohan
authors.initials: J
authors.lastname: Singh
mesh_terms: D001003:Anal Canal / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D009129:Muscle Tonus / Q000502:physiology*; D043263:Myosin-Light-Chain Phosphatase / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; C066737:RHOA protein, human; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein