Decrease in hyperosmotic stress–induced corneal epithelial cell apoptosis by L-carnitine

PURPOSE To characterize the osmoprotective properties of L-carnitine on human corneal epithelial cell volume and apoptosis during hyperosmotic stress. METHODS Human corneal limbal epithelial (HCLE) cells were exposed to culture medium at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) with or without L-carnitine (10 mM). Induction of apoptosis was detected by quantifying the proteolytic activity of caspase-8, caspase-9, and caspase-3/7 using caspase activity assays, the expression of tumor necrosis factor (TNF)-α with enzyme-linked immunosorbent assay, and annexin V/propidium iodide staining of HCLE cells evaluated with confocal microscopy and flow cytometry. Cell volume changes in response to hyperosmotic stress were analyzed using flow cytometry. RESULTS After the HCLE cells were exposed to hyperosmotic medium (500 mOsm), the percentage of shrunken cells and damaged/dead cells (stained positively for annexin V and/or propidium iodide) was six- and three-fold, respectively, higher than that under isotonic conditions (300 mOsm). This was paralleled by an increase in TNF-α concentration in media and caspase-8, -9, and -3/7 activities (six-, four-, ten-, and twelve-fold, respectively; all showing p < 0.001). Addition of L-carnitine during hyperosmotic stress partly restored cell volume and significantly reduced the concentration of TNF-α released (p = 0.005) and caspase-9 activity (p = 0.0125). Addition of L-carnitine reduced the percentage of hyperosmolarity-induced damaged/dead cells to levels observed under isotonic conditions. CONCLUSIONS L-carnitine can regulate human corneal epithelial cell volume under hyperosmotic stress and ameliorate hyperosmotic stress-induced apoptosis.