Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells.
OBJECTIVE
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.
Ramachandran
Charanya
C
School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Satpathy
Minati
M
Mehta
Dolly
D
Srinivas
Sangly P
SP
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1 / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D018994:Myosin Light Chains / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D019332:Endothelin-1; D018994:Myosin Light Chains; C066737:RHOA protein, human; D005576:Colforsin; D000242:Cyclic AMP; D020742:rhoA GTP-Binding Protein; D015739:Nocodazole