Effect of norepinephrine on proliferation, migration, and adhesion of SV-40 transformed human corneal epithelial cells

Christopher J Murphy, Sean Campbell, Kaoru Araki-Sasaki, Carl F. Marfurt

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

Purpose. 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 μM. 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, 25x103 cells were plated onto FNC-coated 96-well plates, incubated in 10 nM- 100 μM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-μm pore size and FNC-coated filters. Cells (250x103) 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 celt 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.

Original languageEnglish (US)
Pages (from-to)529-536
Number of pages8
JournalCornea
Volume17
Issue number5
StatePublished - 1998
Externally publishedYes

Keywords

  • Catecholamines
  • Cell adhesion
  • Cell migration
  • Cell proliferation
  • Cornea
  • Corneal epithelium
  • Corneal innervation
  • Neurotrophic
  • Norepinephrine

ASJC Scopus subject areas

  • Ophthalmology

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