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S.Y. Ghoghawala, C.E. Pullar, M.J. Mannis, R.R. Isseroff; Beta–Adrenergic Receptor (AR) Antagonists Accelerate Corneal Wound Healing in vitro, While Beta–AR Agonists Slow Cell Migration . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5032.
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© ARVO (1962-2015); The Authors (2016-present)
ß–Adrenergic Receptor (AR) antagonists, such as timolol, are commonly prescribed for the treatment of glaucoma, and their effects on corneal wound healing have been evaluated, with conflicting results. We further investigated the effects of ß–AR antagonists and agonists on corneal epithelial wound healing in vitro and their mechanism of action.
Scratch Wound Assay: After growing adult human corneal epithelial (AHCE) cells to confluence, 1–mm wide scratch wounds were made, culture medium was replaced with corneal growth medium (CGM), CGM with 20 µM timolol or CGM with 10 nM isoproterenol, and the area of the wound was monitored microscopically. Immunoblotting: AHCE cells were incubated in either CGM, CGM with 20 µM timolol or CGM with 10 nM isoproterenol and their lysates were electrophoresed, transferred to membranes, and immunoblotted with anti–extracellular signal–related kinase (ERK) and phospho–ERK antibodies. Single Cell Migration Assay: AHCE cells were pre–incubated with CGM +/– okadaic acid (OA), an inhibitor of phosphatase PP2A. At time 0, untreated cells received CGM, CGM with 20 µM timolol or CGM with 10 nM isoproterenol, while OA pretreated cells received either CGM with 100 nM OA or CGM with 100 nM OA+10 nM isoproterenol. Cell migration was monitored microscopically.
Scratch Wound Assay: ß–AR antagonist treated wounds healed completely by 20 hrs, while healing was decreased in control (60%) and agonist treated wounds (16%). Immunoblotting: A ß–AR antagonist increased (10–fold), while agonist decreased (0.6–fold), ERK phosphorylation compared to basal, untreated values. Single Cell Migration Assay: A ß–AR agonist significantly decreased (34%), while antagonist significantly increased (60%), the AHCE cell migratory speed. Pretreatment with 100 nM OA alone had no effect but prevented the ß–AR agonist–mediated decrease in the rate of cell migration.
ß–AR antagonists and agonists have opposite effects on AHCE cell migration, the healing of scratch wounds and the phosphorylation of the promigratory ERK signal. Antagonists increase cell migratory speed, wound healing and ERK phosphorylation, while agonists decrease these parameters. Modulation of the phosphatase PP2A activity by ß–AR agonists mediates the noted decrease in ERK phosphorylation as well as the decrease in migratory speed, as demonstrated by the reversal of the migratory inhibition of isoproterenol by pretreatment with OA at a concentration highly selective for PP2A.
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