Benzalkonium chloride induces dephosphorylation of Myosin light chain in cultured corneal epithelial cells.
OBJECTIVE
Phosphorylation of myosin light chain (MLC) is essential for the contractility of the actin cytoskeleton, which regulates barrier integrity, adhesion, and migration. This study was conducted to investigate the effect of benzalkonium chloride (BAK), a preservative in topical ophthalmic formulations, on MLC phosphorylation in primary cultures of bovine corneal epithelial cells (BCECs).


METHODS
MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by Western blot analysis. Activation of RhoA, which inhibits MLC phosphatase through Rho kinase, was examined by immunoprecipitation. The release of adenosine triphosphate (ATP) was measured by the luciferase-luciferin bioluminescence technique.


RESULTS
Positive expression of MLC kinase (MLCK) was found at the mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Exposure to BAK for 10 to 20 minutes at concentrations of 0.0005%, 0.001%, and 0.003% reduced MLC phosphorylation by more than 30%. In addition, BAK led to thinning of the cortical actin and a decrease in cell adhesion. However, RhoA activity was found to increase with BAK treatment. Similar to BAK, ATP-depletion (induced by both antimycin-A and hypoxia) led to MLC dephosphorylation. BAK exposure also showed acute ATP release.


CONCLUSIONS
BAK induces acute ATP release and concomitant MLC dephosphorylation in bovine corneal epithelial cells. The dephosphorylation, presumably due to ATP loss, is indicative of a loss of contractility of the actin cytoskeleton that could affect cellular functions contributing to the maintenance of epithelial barrier integrity.
Guo
Ying
Y
School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Satpathy
Minati
M
Wilson
Graeme
G
Srinivas
Sangly P
SP
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001548:Benzalkonium Compounds / Q000494:pharmacology*; D015153:Blotting, Western; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D000890:Anti-Infective Agents; D001548:Benzalkonium Compounds; D018994:Myosin Light Chains; D011310:Preservatives, Pharmaceutical; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D051606:G-Protein-Coupled Receptor Kinase 1; D009219:Myosin-Light-Chain Kinase