Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells.
OBJECTIVE
Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells.


METHODS
Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa).


RESULTS
RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632.


CONCLUSIONS
Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.
Satpathy
M
M
School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405, USA.
Gallagher
P
P
Lizotte-Waniewski
M
M
Srinivas
S P
SP
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects; D004728:Endothelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000187:drug effects / Q000378:metabolism*; D024462:Myosin Type II / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D044463:Receptor, PAR-1; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D024462:Myosin Type II; D020742:rhoA GTP-Binding Protein; D002118:Calcium