Regulation of Endothelin-1-Induced Trabecular Meshwork Cell Contractility by Latanoprostene Bunod

Invest Ophthalmol Vis Sci. 2015 Jun;56(6):4108-16. doi: 10.1167/iovs.14-16015.

Abstract

Purpose: Previous in vivo studies demonstrated that latanoprostene bunod (LBN), a nitric oxide (NO)-donating prostaglandin F2α receptor agonist, results in greater intraocular pressure (IOP) lowering than latanoprost. The present series of investigations compared the effects of LBN and latanoprost on primary human trabecular meshwork cell (HTMC) contractility and underlying signaling pathways to determine whether LBN might mediate this additional IOP lowering via the conventional outflow pathway.

Methods: The effect of LBN (1-100 μM) on HTMC cGMP levels was determined by ELISA with or without the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Endothelin-1 (ET-1) was used to induce HTMC contractility. To determine the effect of LBN on myosin light chain-2 (MLC-2) phosphorylation, HTMCs were pretreated with 10 to 60 μM LBN for 1 hour and then ET-1 for 5 minutes. MLC-2 phosphorylation was determined by Western blotting. Effects of LBN (30 and 45 μM) on ET-1-induced filamentous (F)-actin cytoskeletal stress fibers and the focal adhesion associated protein vinculin were determined by confocal microscopy. ET-1-induced HTMC monolayer resistance in the presence of LBN (45 μM) was determined by electrical cell substrate impedance sensing, as an indicator of cell contractility. Latanoprost and SE 175 (an NO donor which releases NO on reductive transformation within the cells) were used as comparators in all studies.

Results: LBN (1-100 μM) significantly increased cGMP levels in a dose-dependent manner, with a half maximal effective concentration (EC50) of 1.5 ± 1.3 μM, and with maximal effect similar to that of 100 μM SE 175. In contrast, latanoprost caused a minimal increase in cGMP levels at 100 μM only. The cGMP elevation induced by LBN or SE 175 was abolished by ODQ and was therefore sGC-dependent. The two NO donors SE 175 and LBN elicited a reduction in ET-1-induced MLC-2 phosphorylation that was significantly greater than that mediated by latanoprost in HTMCs. SE 175 (100 μM) and LBN (30 or 45 μM) caused a dramatic reduction in ET-1-induced actin stress fibers and vinculin localization at focal adhesions, whereas 45 μM latanoprost was without observable effect. SE 175 reduced ET-1-induced increases in HTMC resistance in a dose-dependent manner. A synergistic effect on reduction of HTMC resistance was observed when latanoprost and SE 175 doses were given together. LBN significantly reduced ET-1-induced HTMC monolayer resistance increases to a greater extent than latanoprost, indicating a greater reduction in cell contractility with LBN.

Conclusions: LBN, SE 175, and latanoprost caused relaxation of ET-1-contracted HTMCs. The effect on HTMC relaxation observed with LBN was significantly greater in magnitude than that observed with latanoprost or SE 175. Data indicate that the NO-donating moiety of LBN mediates HTMC relaxation through activation of the cGMP signaling pathway and a subsequent reduction in MLC-2 phosphorylation. These findings suggest that increased conventional outflow facility may mediate the additional IOP-lowering effects of LBN over that of latanoprost observed in in vivo studies.

MeSH terms

  • Antihypertensive Agents / pharmacology*
  • Cell Adhesion / drug effects
  • Cell Physiological Phenomena / drug effects*
  • Cells, Cultured
  • Cyclic GMP / metabolism
  • Cytoskeleton / drug effects
  • Electrophysiological Phenomena / drug effects
  • Endothelin-1 / pharmacology
  • Humans
  • Intraocular Pressure / drug effects
  • Latanoprost
  • Nitric Oxide / metabolism
  • Nitric Oxide Donors / pharmacology*
  • Prostaglandins F, Synthetic / pharmacology*
  • Receptors, Prostaglandin / antagonists & inhibitors
  • Trabecular Meshwork / drug effects*

Substances

  • Antihypertensive Agents
  • Endothelin-1
  • Nitric Oxide Donors
  • Prostaglandins F, Synthetic
  • Receptors, Prostaglandin
  • Nitric Oxide
  • Latanoprost
  • Cyclic GMP