Effect of norepinephrine on proliferation, migration, and adhesion of SV-40 transformed human corneal epithelial cells.
OBJECTIVE
To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial 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 concentrations of 100 nM-100 microM. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25 x 10(-3) cells were plated onto FNC-coated 96-well plates, incubated in 10 nM-100 microM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-microm pore size and FNC-coated filters. Cells (250 x 10(3)) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard 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 migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested.


CONCLUSIONS
Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.
Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 53706, USA.
C J
CJ
Murphy
S
S
Campbell
K
K
Araki-Sasaki
C F
CF
Marfurt
D000952:Antigens, Polyomavirus Transforming; D002448:Cell Adhesion / Q000187:drug effects*; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects*; D002461:Cell Line, Transformed / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D003315:Cornea / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D009638:Norepinephrine / Q000494:pharmacology*; D013539:Simian virus 40
D000952:Antigens, Polyomavirus Transforming; D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; D009638:Norepinephrine