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 p(Thr696)-MYPT1, 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 concentration-dependent 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 (Gö 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 (MLC(20)), phospho-MYPT1, and phospho-MLC(20) 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.
Patel
Chirag A
CA
Dept. of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson Univ., 1025 Walnut St., Rm. 901 College, Philadelphia, PA 19107, USA.
Rattan
Satish
S
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D001003:Anal Canal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D009126:Muscle Relaxation / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012007:Rectum / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000577:Amides; D002352:Carrier Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D008301:Maleimides; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein; C070515:bisindolylmaleimide I