title: Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A.
abstract: OBJECTIVE
  Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may represent a new way of influencing outflow facility through isolated relaxation of the trabecular meshwork (TM). This work was performed to investigate the existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GTPase)-mediated pathway.
  
  
  METHODS
  Isometric tension measurements of bovine TM and ciliary muscle (CM) were performed. Intra- and extracellular calcium buffering was accomplished with EGTA and 1, 2-bis(2-aminophenoxy)-ethane-N,N:,N:,N:',N:'-tetra-acetic acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4alpha-phorbol. Calcium-independent contraction was blocked using the highly specific ROCK inhibitor Y-27632. Western blot analysis and immunoprecipitation was performed using human TM cells.
  
  
  RESULTS
  In TM, carbachol induced partial contraction under conditions of extracellular calcium depletion (22. 1% +/- 2.3% versus 100%, n = 9). The membrane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%, n = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 9) but not in CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4alpha-phorbol did not induce contraction, indicating that activation of PKC is involved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein in human TM cells.
  
  
  CONCLUSIONS
  The data indicate that contrary to CM, the TM features calcium-independent contractile mechanisms linked to rho-A and PKC isoforms that do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.
authors.affiliation: Institut für Klinische Physiologie, and. Augenklinik Universitätsklinikum Benjamin Franklin, Freie Universität Berlin. Klinik und Poliklinik für Augenheilkunde, Charitè, Campus Virchow Klinikum, Humboldt Universität Berlin, German.
authors.forename: H
authors.initials: H
authors.lastname: Thieme
authors.forename: M
authors.initials: M
authors.lastname: Nuskovski
authors.forename: J U
authors.initials: JU
authors.lastname: Nass
authors.forename: U
authors.initials: U
authors.lastname: Pleyer
authors.forename: O
authors.initials: O
authors.lastname: Strauss
authors.forename: M
authors.initials: M
authors.lastname: Wiederholt
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D002924:Ciliary Body / Q000187:drug effects / Q000201:enzymology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004594:Electrophysiology; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007537:Isometric Contraction; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology*; D010704:Phorbols / Q000494:pharmacology; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014129:Trabecular Meshwork / Q000187:drug effects / Q000201:enzymology*; D054460:rho-Associated Kinases
chemical_list: D000577:Amides; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D010704:Phorbols; D011725:Pyridines; C108830:Y 27632; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C033085:phorbol