title: Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells.
abstract: Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). We determined the effects of PDBu (10(-7) M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. We used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10(-6) M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10(-6) M). We also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined.
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Jagmohan
authors.initials: J
authors.lastname: Singh
authors.forename: Pinckney J
authors.initials: PJ
authors.lastname: Maxwell
authors.forename: Satish
authors.initials: S
authors.lastname: Rattan
mesh_terms: D036002:ADP Ribose Transferases / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D001003:Anal Canal / Q000378:metabolism; D001905:Botulinum Toxins / Q000494:pharmacology; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006801:Humans; D009119:Muscle Contraction / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000577:Amides; D009281:Naphthalenes; D011725:Pyridines; C066737:RHOA protein, human; C108830:Y 27632; D015240:Phorbol 12,13-Dibutyrate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D054460:rho-Associated Kinases; D051571:Protein Kinase C-alpha; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein; C058819:calphostin C