EGF suppresses hydrogen peroxide induced Ca 2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells

Abstract

Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca 2+] i ; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca 2+] i . Whereas pathophysiological concentrations of H 2O 2 (10 m m) induced irreversible large increases in [Ca 2+] i , lower concentrations (up to 1 m m) had smaller effects, which were further reduced by exposure to either 5 μ m nifedipine or EGF (10 ng ml −1). EGF had a larger protective effect against H 2O 2-induced rises in [Ca 2+] i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 μ m) on [Ca 2+] i . At 10 μ m, it reversibly elevated [Ca 2+] i whereas at 50 μ m an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H 2O 2 induces rises in [Ca 2+] i that occur through increases in Ca 2+ permeation along plasma membrane pathways that include L-type Ca 2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H 2O 2-induced rises in [Ca 2+] i , its presence during eye bank storage could improve the outcome of corneal transplant surgery.