Effects of osmoprotectants on hyperosmolar stress in cultured human corneal epithelial cells.
OBJECTIVE
Increased tear osmolarity in dry eye disease has been found to stimulate production of inflammatory cytokines and matrix metalloproteinases by ocular surface epithelial cells. Prokaryotic and mammalian organ system cells maintain normal function under hypertonic conditions by the synthesis or accumulation of osmoprotectant compounds. This study assessed the effect of osmoprotectant compounds on the activation state of mitogen-activated protein (MAP) kinases in human corneal epithelial cells incubated in hyperosmolar conditions.


METHODS
Human corneal epithelial cells were incubated in media of isotonic, physiological osmolarity (300 mOsm) and in hyperosmolar media (400 mOsm), in the presence and absence of osmoprotectants, including several amino acids (L-carnitine and betaine), glycerol, and the polyol erythritol. The phosphorylation (activation) states of c-Jun N-terminal kinases (JNK) and p38 MAP kinases were monitored by Western blot and bead-based immunoassays.


RESULTS
Hyperosmolar conditions achieved by addition of sodium chloride or sucrose increased ratios of phosphorylated JNK and p38 to total JNK and p38. Compared with controls, 10 mM L-carnitine or 40 mM erythritol significantly lowered levels of activated MAP kinases in response to hyperosmolar stress. They also lowered ratios of phosphorylated to total kinases to barely detectable levels in cells cultured in isotonic media.


CONCLUSIONS
The osmoprotectants L-carnitine and erythritol, alone or in combination, were found to protect against stress activation of corneal epithelial cells cultured in hyperosmolar media.
Corrales
Rosa M
RM
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030, USA.
Luo
Lihui
L
Chang
Eliseu Y
EY
Pflugfelder
Stephen C
SC
D015153:Blotting, Western; D002331:Carnitine / Q000494:pharmacology*; D002470:Cell Survival; D002478:Cells, Cultured; D019610:Cytoprotection; D004359:Drug Therapy, Combination; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D004896:Erythritol / Q000494:pharmacology*; D006801:Humans; D006982:Hypertonic Solutions / Q000633:toxicity; D007552:Isotonic Solutions / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D009883:Ophthalmic Solutions / Q000494:pharmacology*; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010766:Phosphorylation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D006982:Hypertonic Solutions; D007552:Isotonic Solutions; D009883:Ophthalmic Solutions; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D004896:Erythritol; D002331:Carnitine