EFFECT OF NOREPINEPHRINE ON PROLIFERATION, MIGRATION, AND ADHESION OFSV40-TRANSFORMED HUMAN CORNEAL EPITHELIAL-CELLS

Purpose: To determine the ability of norepinephrine to modulate prolif eration, adhesion, and migration of SV-40 transformed human corneal ep ithelial cells. Methods. Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose response curve was generated for norepinephrine in concentration s of 100 nM-100 mu M. The cell number in each well was evaluated using the fluorochrome Calcein AM tan intracellular esterase cleavage subst rate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25x10(3) cells were plated onto FNC-c oated 96-well plates, incubated in 10 nM-100 mu M norepinephrine for 9 0 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-mu m pore size and FNC-coated filters. Cells (250x10(3)) were add ed to the upper chamber, incubated for Is h in the presence of factors , after which rime the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a sta ndard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed. Results. Norepinephrine stimulated corneal epithelial cell proliferation and m igration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphy siologic) concentration tested. Conclusions. Norepinephrine is normall y found in the cornea and may be important in the maintenance of norma l corneal homeostasis and in wound-healing processes.