Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells.
Increased contractility of the actin cytoskeleton by phosphorylation of the regulatory myosin light chain (MLC) results in a loss of barrier integrity in corneal endothelial cells. This study has investigated the effect of extracellular ATP, which may influence both Ca2+ and cAMP signalling, on MLC phosphorylation and barrier integrity in cultured bovine corneal endothelial cells (BCEC) known to express A2B and P2Y purinergic receptors, and ecto-nucleotidases. Extracellular ATP (100 microM) promoted MLC dephosphorylation (pMLC=61.8% at 18 min; n=9). Pre-exposure to ARL-67156, an ecto-nucleotidase inhibitor, prevented ATP-induced dephosphorylation. Other P2Y agonists, UTP and ATPgammaS, also induced MLC dephosphorylation but to a lesser degree compared to ATP. Thrombin (2 U/ml), which activate Rho kinase through PAR-1 receptors in the endothelium, induced MLC phosphorylation (pMLC=129.2%; n=14). This phosphorylation was completely abolished by concomitant exposure to ATP. When cells were pretreated with adenosine (100 microM; A2B agonist) or forskolin (10 microM), thrombin-induced phosphorylation was suppressed. ATP also led to a significant increase in cAMP (> 3-fold compared to 10 microM adenosine). Thrombin-induced increase in trans-endothelial flux of horseradish peroxidase (44 kDa) and disruption of the cortical actin were suppressed by ATP. These findings indicate that in BCEC (1) ATP induces elevated cAMP through its metabolite adenosine leading to MLC dephosphorylation, (2) Stimulation of P2Y2 receptors also leads to activation of MLCP since UTP- and ATPgammaS caused MLC dephosphorylation, and (3) ATP is antagonistic to thrombin since the latter inhibits MLCP through increased activity of Rho kinase. These findings further emphasize the role of contractility of the actin cytoskeleton in regulating the barrier integrity of corneal endothelium.
Satpathy
M
M
School of Optometry, Indiana University, 800 East Atwater Avenue, Indiana University, Bloomington, IN 47405, USA.
Gallagher
P
P
Jin
Y
Y
Srinivas
S P
SP
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis; D004728:Endothelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D058910:Purinergic P2 Receptor Agonists; D058473:Receptors, Purinergic P2Y2; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000494:pharmacology
C092431:6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP; D000199:Actins; D018994:Myosin Light Chains; D058910:Purinergic P2 Receptor Agonists; D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D013917:Thrombin