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Elliot D. Cha, Haiyan Gong; Comparative Studies of Rho/Rock Pathway of Bovine Eyes After Y-27632 Treatment or Induction of Washout. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4658.
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Our previous studies showed that increased outflow facility (C) by treatment with the Rho kinase inhibitor, Y-27632 (Y27), for 30 minutes and induction of washout, by 3 hours perfusion without an inhibitor, showed similar morphological changes in bovine eyes. The current study is aimed at determining if these two treatments employ similar regulatory mechanisms to increase C by reducing phosphorylation of myosin light chain 2 (MLC2) through inhibition of the Rho/ROCK pathway. We hypothesize that both washout and Y27 will cause a decrease in phosphorylation of MLC2.
12 freshly enucleated pairs of bovine eyes were first perfused with dulbecco’s PBS with 5.5mM glucose (GPBS) for 30 minutes to establish a stable baseline C. Eyes were then treated either by short term (30 minutes) GPBS (N=6) or 50µM Y27 (N=6) or long term (3 hours) GPBS (N=6) or 50µM Y27 (N=6). Normal non-perfused eyes were the control. Following perfusion, whole eyes were processed for either western immunoblotting or confocal immunohistochemistry. Both technologies probed for phospho-MLC2. Student’s t-test (p≤0.05) was used for statistical analysis.
Perfusion data revealed that C increased significantly from baseline in short and long term treatments with both Y27 and GPBS. No significant difference in C was found between short term treatments; however, C was significantly higher after 3hours Y27 compared to 3hours GPBS (washout). This difference is believed to be a result of an additive effect during long term perfusion of the Y27 solution (Y27 + GPBS). Western immunoblots revealed that all treatments reduced phosphorylation of MLC2 except long term GPBS, which showed an increase in phosphorylation. This suggests that the increased C by washout is regulated through an alternative pathway. Samples probed for phospho-MLC2 in confocal immunohistochemistry revealed punctate staining in the trabecular meshwork of untreated eyes and a reduction in fluorescent staining by all treatments except long term GPBS, where an increase was observed.
Although both Y27 and washout caused an increase in C, both immunoblotting and immunohistochemistry suggests that washout increases phosphorylation and Y27 decreases it. This variation leads us to believe that the two treatments utilize different regulatory mechanisms to increase C and the increased C in washout is not contingent on the reduction of phosphorylated MLC2 through inhibition of the Rho/ROCK pathway. Therefore, further understanding of the regulatory mechanism(s) underlying washout may provide a means to an alternative therapy to reduce IOP in human eyes with glaucoma.
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