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Diana Santander-Garcia, Beatriz Marcos-Ramiro, Susana Barroso, Ignacio Jimenez-Alfaro, Jaime Millan; Hyperosmotic shock activates RhoA and Rac1 in corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4905.
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© ARVO (1962-2015); The Authors (2016-present)
To study the involvement of Rho GTPase-mediated pathways in the corneal endothelial barrier response to hypertonic stress.
Experiments were performed in human endothelial cell line HCEC B4G12 in vitro. After exposure to hypertonic stress, the activity of the Rho GTPases RhoA, Rac1 and Cdc42 was analyzed by pulldown assay. The activation of downstream effectors of Rho was detected by western blot. Electric Cell-Substrate Impedance Sensing (ECIS) of endothelial monolayers was measured to assess the influence of osmotic stress on barrier function. Endothelial barrier integrity was also analyzed by immunofluorescence and confocal microscopy. To determine the relative contribution of RhoA- and Rac1 mediated pathways to the corneal endothelial responses, we tested the effect of Y-27632, the inhibitor of the RhoA effector Rho kinase (ROCK), and of EHop-016, a new inhibitor of Rac1, on endothelial barrier disruption in response to hypertonic stress.
Exposure to NaCl (487 mM; 10 min) generated cell contraction, which was detected by a 60% decrease in transendothelial electrical resistance. NaCl induced the activation of both RhoA (p=0, 0441) and Rac1 (p= 0,0437), but not of Cdc42. Phosphorylation of myosin light chain and ezrin-radixin-moesin proteins, which are activated downstream RhoA was also induced 2 fold (p=0.08) and 1.67 fold (p=0.043), respectively. Incubation with the ROCK inhibitor Y-27632 (5 microM) attenuated resistance decrease upon NaCl exposure and delayed barrier recovery after stress withdrawal. In contrast, EHop-016 (1 microM) did not produce any change in the barrier response to stress.
Both RhoA and Rac1 signalling are activated during the contraction of human corneal endothelial cells after exposure to hypertonicity. ROCK signalling inhibition by Y-27632 reduces the dynamics of barrier remodeling upon exposure and withdrawal of hyperosmotic stress.
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