advertisement
Abstract #20739 Published in IGR 10-1
Forskolin induces Myosin light chain dephosphorylation in bovine trabecular meshwork cells
Ramachandran C; Satpathy M; Mehta D; Srinivas SPCurrent Eye Research 2008; 33: 169-176
PURPOSE: Enhanced contractility of the actin cytoskeleton in trabecular meshwork (TM) cells is implicated in increased resistance to aqueous humor outflow. In this study, we have investigated effects of forskolin, which is known to elevate cAMP and also enhance aqueous humor outflow, on myosin light chain (MLC) phosphorylation, a biochemical marker of actin contractility. METHODS: Experiments were performed using cultured bovine TM cells. Phosphorylated MLC (pMLC), expressed as the % of untreated cells, was assessed by urea-glycerol gel electrophoresis and Western blotting. RhoA activity was determined by affinity precipitation of RhoA-GTP to RhoA binding domain of an effector of RhoA. Intracellular cAMP levels were measured by ELISA. RESULTS: Exposure to LPA (lysophosphatidic acid) led to increased MLC phosphorylation (LPA: pMLC = 133%) and activation of RhoA. These responses of LPA were suppressed by co-treatment with forskolin (LPA + forskolin: pMLC= 88%). Similarly, ET-1 and nocodazole-induced MLC phosphorylation (ET-1: pMLC = 145%; nocodazole: pMLC = 145%) as well as RhoA activation were suppressed by co-treatment with forskolin (ET-1 + forskolin: pMLC = 99%; nocodazole + forskolin: pMLC = 107%). Exposure to forskolin alone led to MLC dephosphorylation (pMLC = 68%). Forskolin alone led to a 4-fold increase in cAMP levels. This increase was not affected when co-treated with LPA or ET-1. CONCLUSIONS: Forskolin prevents MLC phosphorylation induced by LPA, ET-1, and nocodazole through inhibition of RhoA-Rho kinase axis. MLC dephosphorylation and consequent relaxation of actin cytoskeleton in TM cells presumably underlies the increased outflow facility reported in response to forskolin.
Dr. C. Ramachandran, School of Optometry, Indiana University, Bloomington, Indiana, USA
Classification:
3.8 Pharmacology (Part of: 3 Laboratory methods)
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)
