title: Concentration-related effects of nitric oxide and endothelin-1 on human trabecular meshwork cell contractility.
abstract: The contractility status of trabecular meshwork (TM) cells influences aqueous humor outflow resistance and intraocular pressure. Using human TM cells as a model, the goal of the present study was to examine concentration-response relationships of two prototypical molecules, nitric oxide (NO) and endothelin-1 (ET-1), known to differentially influence vascular smooth muscle contractility. Efficacy of ET-1, two NO donors (DETA-NO and SNP) and a cGMP analog (8-Br-cGMP) were assessed using two complementary methods: functionally in a gel contraction assay and biochemically using a myosin light chain phosphorylation assay. The NO donors DETA-NO and SNP dose dependently relaxed cultured human TM cells (EC50 for DETA-NO = 6.0 ± 2.4 μM, SNP = 12.6 ± 8.8 μM), with maximum effects at 100 μM. Interestingly, at concentrations of NO donors above 100 μM, the relaxing effect was lost. Relaxation caused by DETA-NO (100 μM) was dose dependently blocked by the soluble guanylate cyclase specific inhibitor ODQ (IC50 = 460 ± 190 nM). In contrast to the NO donors, treatment of cells with the cGMP analog, 8-Br-cGMP produced the largest relaxation (109.4%) that persisted at high concentrations (EC50 = 110 ± 40 μM). ET-1 caused a dose-dependent contraction of human TM cells (EC50 = 1.5 ± 0.5 pM), with maximum effect at 100 pM (56.1%) and this contraction was reversed by DETA-NO (100 μM). Consistent with functional data, phosphorylation status of myosin light chain was dose dependently reduced with DETA-NO, and increased with ET-1. Together, data show that TM cells rapidly change their contractility status over a wide dynamic range, well suited for the regulation of outflow resistance and intraocular pressure.
authors.affiliation: Department of Ophthalmology, Duke University, Durham, NC, USA.
authors.forename: W Michael
authors.initials: WM
authors.lastname: Dismuke
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, NC, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Liang
authors.affiliation: Department of Bioengineering, Imperial College London, London, UK.
authors.forename: Darryl R
authors.initials: DR
authors.lastname: Overby
authors.affiliation: Department of Ophthalmology, Duke University, Durham, NC, USA; Department of Biomedical Engineering, Duke University, Durham, NC, USA. Electronic address: dan.stamer@duke.edu.
authors.forename: W Daniel
authors.initials: WD
authors.lastname: Stamer
mesh_terms: D000328:Adult; D002468:Cell Physiological Phenomena / Q000502:physiology*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology*; D004791:Enzyme Inhibitors; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D006801:Humans; D007223:Infant; D008875:Middle Aged; D018994:Myosin Light Chains / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D010766:Phosphorylation; D011810:Quinoxalines / Q000494:pharmacology; D014019:Tissue Donors; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000502:physiology; D014226:Triazenes / Q000494:pharmacology
chemical_list: C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D020030:Nitric Oxide Donors; D010069:Oxadiazoles; D011810:Quinoxalines; D014226:Triazenes; D009599:Nitroprusside; C016276:8-bromocyclic GMP; D006162:Guanylate Cyclase; D006152:Cyclic GMP
keywords: Schlemm's canal; aqueous humor; conventional outflow; glaucoma