Effects of Endothelin-1 and Flunarizine on Human Trabecular Meshwork Cell Contraction

HTM Cell Culture Model System.

HTM cell cultures were established from normotensive donor eyes. The globes were soaked in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) plus 5% penicillin/streptomycin solution and 1% l-glutamine, then bifurcated at the equator. The TM area was isolated under the dissecting microscope at ×50 magnification with a micro scalpel, grasped with forceps, and removed, taking care to exclude the Schlemm’s canal portion.

The HTM cells were cultured in 6-well plates at 37°C in a 5% CO₂ atmosphere in the same medium; confluence was reached after 3 weeks. HTM cells from the established three primary lines were characterized with respect to their growth characteristics, morphology, and cytoskeletal proteins: the immunocytochemical procedures for detection of smooth-muscle actin (antisera purchased from DakoCytomation, Glostrup, Denmark) were performed as described elsewhere (10). HTM cell viability was checked with trypan blue staining.

Low-confluence HTM cell cultures at the first or second passage were used. All experiments were performed in triplicate wells and repeated at least two independent times using cells from the three established cell lines.

For each set of experiments, a concentration of 2000 cells/ml culture medium was used, and HTM cells were set in 24-well plates.

Effect of ET - 1 on HTM Cells.

ET - 1 (C₁₀₉H₁₅₉N₂₅O₃₂S₅, batch No. L02049/e) was purchased from Alexis Biochemicals, Lausen, Switzerland, and stored at −20°C until used.

A 20 μl aliquot of 10 μM endothelin dissolved in 0.01 M phosphate-buffered saline (PBS), pH 7.2, was added to the medium and administered to the cells, wells were carefully shaken, and the plates were set in the CO₂ incubator. As a control, 20 μl PBS was added to the medium of control wells. Cells at baseline, 5 mins, and 10 mins after the addition of ET-1 or PBS were observed with a Zeiss Axiovert inverted microscope, and digitized micrographs of the cells were acquired with the Nikon Coolpix 990 camera.

To characterize the mechanisms by which ET-1 stimulated contractile force generation, we added 10 μM ET-1 to cells treated with 10 μM flunarizine (a calcium channel blocker that affects vessel calcium l-channels and n-channels at a neuronal level; Ref. 11). Cells at baseline and 5 mins and 10 mins after the addition of flunarizine plus ET-1 were observed, and micrographs were again recorded.

Cell Contraction and Image Analysis.

The contractile status of the cells was evaluated by a morphometric analysis of cell area, assuming that, as previously demonstrated (12, 13), HTM cells in tissue culture are able to reduce their area as a consequence of cytoskeletal contraction rather than regulatory volume decrease.

Image analysis was performed on the computer-acquired digitized micrographs taken in all of the experiments using image-processing software (Image Pro Plus, version 3.0.2; Media Cybernetics, Silver Spring, MD).

The morphometric evaluation of the cell area was semi-automatically performed on a cell-to-cell basis by overlying the cell periphery with the digital cursor. A total of 250 cells were measured for each experiment. Statistical evaluations were performed with the Statistical Package for Social Science (SPSS) program package (SPSS Inc., Chicago, IL) using an unpaired Student’s t test; values of P < 0.05 were regarded as statistically significant.