Abstract
Abstract: :
Purpose: It has been shown that substance P (SP) will stimulate corneal wound healing and epithelial cell migration, however, this occurs at very high concentrations of SP (approximately 200 to 400 uM). We therefore investigated the ability of different concentrations of SP to stimulate calcium release in a human corneal epithelial cell to see how this correlated with the dose required for wound healing. Methods: Different concentration of SP were added to human corneal epithelial cells (SV–40 HCE) grown to confluency on glass coverslips and then incubated with Fura–2AM. These were then placed in a cuvette in CSB buffer. The fluorescence was then measured at 510nm in a PTI spectrophotometer to determine the amount free Calcium versus time. Different combinations of growth factors were flowed through the cuvette at 3 ml/min. Results: The dose response curve for the SP stimulation of the cornea epithelial cells showed an EC50 of approximately 200 uM. SP was also found to be synergistic with IGF at 25 ng/ml, EGF at 25 ng/ml and Bactenecin (bovine L–defensin) at 100 ng/ml. In looking for the possible involvement of other receptors in this process we tested BTX and tubocuarine. We found no inhibition with BTX at 1 uM and slight inhibition with tubocuarine at 500 ng/ml. Conclusions: The concentration of SP required for calcium release is consistent with that used by SP for corneal wound healing and epithelial cell migration. However, the high concentration of SP required to stimulate calcium release in human corneal epithelial cells is not consistent with SP using the classical NK–1 receptor.
Keywords: cornea: epithelium • cornea: basic science • wound healing