Abstract
Abstract: :
Purpose: Increased contractility of the actin cytoskeleton is implicated in enhanced resistance to aqueous humor outflow across the trabecular meshwork (TM). A principal determinant of contractility is the phosphorylation status of the regulatory light chain of myosin II (MLC). Phosphorylation of MLC, which is promoted by MLC kinase (MLCK), is opposed by MLC phosphatase (MLCP). MLCP inactivation, and hence increased contractility, is induced predominantly by phosphorylation of MYPT1, the regulatory subunit of MLCP, at Thr696 and Thr850 by Rho kinase. This phosphorylation inhibits PPC1Δ, the catalytic domain of MLCP. In this study, we have investigated the effects of elevated cAMP on MLC phosphorylation status in cultured bovine TM cells. Methods: MLC phosphorylation was assessed by urea–glycerol gel electrophoresis and Western blotting. cAMP was elevated by direct activation of adenylate cyclase using forskolin (10 µM).Results: (a) Exposure to forksolin for 10 min led to significant MLC dephosphorylation (%pMLC = 55% of control; n = 6). (b) Forskolin suppressed Y–27632–sensitive MLC phosphorylation induced by Gα12/13–coupled LPA1 agonist (LPA; 10 µM; 10 min) and ETA agonist (ET–1; 100 nM; 10 min). (c) Treatment of cells with nocodazole (2 µM; 30 min), known to disrupt microtubules and activate Rho kinase, led to increased MLC phosphorylation. This increase was suppressed when cells were co–treated with forskolin. (e) Exposure to ML–7 at its saturating concentration (50 µM; 30 min), known to inactivate MLCK, blocked MLC phosphorylation (% pMLC = 77; n = 6) but the effect was significantly smaller compared to forskolin. Conclusions: Elevated cAMP induces relaxation of TM. This is consistent with increased outflow facility by ß2 agonists and forskolin reported with in vitro preparations of the TM. cAMP effect stems largely from inhibition of activation of RhoA–Rho kinase axis since the effects of LPA, ET–1, and nocodazole are all through inhibition of MLCP. Under basal conditions, cAMP is more efficacious in suppressing MLC phosphorylation compared to ML–7 consistent with the effect of the second messenger on inactivation of MLCK as well as activation of MLCP.
Keywords: trabecular meshwork • signal transduction • cytoskeleton