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.

Keywords: Schlemm's canal; aqueous humor; conventional outflow; glaucoma.

Figure 1. Human Trabecular Meshwork Gel Contractility Assay

A) Schematic of experimental design. Collagen gels were cast, human TM cells were seeded onto the gel surface and allowed to attach and grow for one day. Cells were then serum starved in serum-free (SF) medium overnight (18 hrs) before gels were separated from the sides of the well, allowed to stabilize and treated with drug. B) Following detachment of the collagen gel from the side of the well, human TM cells contract the gel in the absence of treatment, reducing the surface area of the gel incrementally for 9 hours (shown schematically in panel A and experimentally in panel B. Data shown were obtained from digitized images of gels (3 different experiments using TM86 and TM 96 cell strains) used to calculate are using Image-J software.

Figure 2. Nitric oxide effects on human TM cell contractility

Shown are concentration-response curves obtained from three different compounds (DETA-NO, SNP and 8-Br-cGMP). In a dose-dependent manner, treatment with the two NO donors DETA-NO and SNP or 8-Br-cGMP increased the area of collagen gels precontracted (without drug) for 9 hours by human TM cells. 8-Br-cGMP was the most efficacious and potent of the three compounds. For 8-Br-cGMP experiments, 2 cell strains (TM86, TM96) were tested in four different experiments. For DETA-NO (0.1–100µM), 4 cell strains (TM86, TM98, TM96, TM93) were examined in 9 different experiments and for (200 and 400µM) 2 cell strains (TM86, TM96) were examined in three different experiments. For SNP (0.1–100µM), 3 cell strains (TM86, TM96, TM93) were evaluated in 4 different experiments and for (1mM) 2 cell strains (TM86, TM93) were tested in three different experiments. Data shown are the mean (±SD).

Figure 3. Soluble guanylate cyclase mediation of nitric oxide-induced relaxation of human TM cells

Human TM cells grown on collagen gels were incubated with the sGC inhibitor, ODQ at increasing concentrations (0.25, 0.5, 1, 5 and 10 µM). After a 5 minute incubation with ODQ, DETA-NO (100µM) was added to media containing ODQ for 5 hours. Changes in gel area resulting from DETA-NO/ODQ treatment were quantified and normalized to untreated controls. Data shown is the mean (±SD) of 4 experiments using 2 different cell strains (TM86, TM96).

Figure 4. Endothelin-1 dose-dependently enhances human TM cell contraction

Recombinant human ET-1 was incubated with human TM cells grown on collagen gels for 5 hours. Changes in gel area resulting from ET-1 treatment were quantified and normalized to untreated controls. Data are the mean (±SD) values obtained from six experiments using 3 cell strains (TM86, TM96, TM93).

Figure 5. Reversal of endothelin-1 induced contraction of human TM cell by the nitric oxide donor DETA-NO

Recombinant human ET-1 (100pM) was incubated with human TM cells grown on collagen gels for 12 hours. In 3 of 6 experiments, DETA-NO (100µM) was added 6 hours after ET-1. Data are mean (±SD). N=3 for ET-1 treated and N=3 for ET-1 + DETA-NO treated. * p<0.05, ET-1 + DETA-NO versus ET-1 only. # p=0.49, 12hr versus initial gel area.

Figure 6. Dose-dependent effects of nitric oxide and endothelin-1 on myosin light chain phosphorylation status in human TM cells

Human TM cell monolayers were treated with increasing concentrations of DETA-NO (panel A) or ET-1 (panel B). Cell lysates were prepared and analyzed in duplicate by SDS-PAGE followed by western blotting using antibodies that recognize either phospho-MLC (blot 1) or total MLC (blot 2). Band intensity was quantified by densitometry, and expressed as a ratio (mean±SD) of p-MLC to total MLC. Blots shown (inset) are representative of results observed for 3 independent experiments using TM 86, TM 96 and TM93 cell strains. * p<0.05 vs no drug

Figure 7. Dynamic range of human TM versus vascular smooth muscle cell contractility

A) Shown are representative images of human TM cells grown on collagen gels and treated with DETA-NO or ET-1 compared to untreated controls. After 5 hours of drug treatment, visible changes in collagen gel area are observable and % changes in the individual gels over time are shown below. B) The results obtained in the present study for human TM cellular contractility (from DETA-NO, SNP and ET-1) are compared to published cellular contractility of smooth muscles cells resulting from ET-1 or NO donors(Bourke et al., 2011; Dallot et al., 2003; Defawe et al., 2010; Kotlo et al., 2011; Lee et al., 1998). All contractions or relaxations shown were results using similar gel contraction assays.