Concentration-related. effects of nitric oxide and endothelin-1 on human trabecular meshwork cell contractility

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 mu M, SNP = 12.6 +/- 8.8 mu M), with maximum effects at 100 mu M. Interestingly, at concentrations of NO donors above 100 mu M, the relaxing effect was lost. Relaxation caused by DETANO (100 mu M) was dose dependently blocked by the soluble guanylate cyclase specific inhibitor ODQ (IC50 = 460 +/- 190 mu M). 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 mu M). ET-1 caused a dose-dependent contraction of human TM cells (EC50 = 1.5 +/- 0.5 mu M), with maximum effect at 100 mu M (56.1%) and this contraction was reversed by DETA-NO (100 pM). 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. (C) 2013 Elsevier Ltd. All rights reserved.