Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A

Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4240-6.

Abstract

Purpose: 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.

MeSH terms

  • Amides / pharmacology
  • Animals
  • Blotting, Western
  • Calcium / antagonists & inhibitors
  • Calcium / pharmacology*
  • Cattle
  • Cells, Cultured
  • Chelating Agents / pharmacology
  • Ciliary Body / drug effects
  • Ciliary Body / enzymology
  • Egtazic Acid / analogs & derivatives*
  • Egtazic Acid / pharmacology
  • Electrophysiology
  • Enzyme Inhibitors
  • Intracellular Signaling Peptides and Proteins
  • Isometric Contraction
  • Muscle Contraction / drug effects
  • Muscle Contraction / physiology*
  • Muscle, Smooth / physiology*
  • Phorbols / pharmacology
  • Precipitin Tests
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism*
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / metabolism*
  • Pyridines / pharmacology
  • Tetradecanoylphorbol Acetate / pharmacology
  • Trabecular Meshwork / drug effects
  • Trabecular Meshwork / enzymology*
  • rho-Associated Kinases

Substances

  • Amides
  • Chelating Agents
  • Enzyme Inhibitors
  • Intracellular Signaling Peptides and Proteins
  • Phorbols
  • Pyridines
  • Y 27632
  • 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • Egtazic Acid
  • Protein Serine-Threonine Kinases
  • rho-Associated Kinases
  • Protein Kinase C
  • Tetradecanoylphorbol Acetate
  • Calcium
  • phorbol