Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells.
OBJECTIVE
To investigate changes in the phosphorylation of myosin light chain (MLC) in response to histamine and its effect on the barrier integrity of corneal epithelial cells.


METHODS
Experiments were performed in bovine corneal epithelial cells (BCEC). RT-PCR and Western blotting were employed to characterize expression of H1 receptors and MLC kinase (MLCK). Phosphorylation of MLC was assessed by urea-glycerol gel electrophoresis and Western blotting. Barrier integrity was determined as permeability to horseradish peroxidase (HRP; 44 kDa) across monolayers grown on porous filters.


RESULTS
Expression of both H1 receptors and MLCK was found in BCEC. Exposure to histamine induced significant MLC phosphorylation concomitant with an increase in HRP permeability. In addition, organization of the cortical actin found in resting cells was disrupted. In contrast to histamine, ATP (a P2Y receptor agonist) induced dephosphorylation of MLC. Pre-exposure to ATP reduced the effect of histamine on HRP permeability and disruption of cortical actin.


CONCLUSIONS
MLC phosphorylation, a biochemical pre-requisite for increased contractility of the actin cytoskeleton, led to histamine-induced breakdown of the barrier integrity in the corneal epithelial cells. This is attributed to weakening of the tethering forces at the tight junctions by the centripetal forces produced by increased actin contractility.
Guo
Ying
Y
School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, Indiana 47405, USA.
Ramachandran
Charanya
C
Satpathy
Minati
M
Srinivas
Sangly P
SP
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006632:Histamine / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D010539:Permeability; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D011969:Receptors, Histamine H1 / Q000235:genetics / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism; D013997:Time Factors; D014544:Uridine Triphosphate / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D007527:Isoenzymes; D018994:Myosin Light Chains; D012333:RNA, Messenger; D011969:Receptors, Histamine H1; D018048:Receptors, Purinergic P2; C022571:adenosine 5'-O-(3-thiotriphosphate); D006632:Histamine; D000255:Adenosine Triphosphate; D006735:Horseradish Peroxidase; D009219:Myosin-Light-Chain Kinase; D020742:rhoA GTP-Binding Protein; D014544:Uridine Triphosphate