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±2.4μM, SNP=12.6±8.8μM), with maximum effects at 100μM. Interestingly, at concentrations of NO donors above 100μM, the relaxing effect was lost. Relaxation caused by DETA-NO (100μM) was dose dependently blocked by the soluble guanylate cyclase specific inhibitor ODQ (IC₅₀=460±190nM). 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±40μM). ET-1 caused a dose-dependent contraction of human TM cells (EC₅₀=1.5±0.5pM), with maximum effect at 100pM (56.1%) and this contraction was reversed by DETA-NO (100μM). 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.