Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and Rho-A

PURPOSE. Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may rep resent a new way of influencing outflow facility through isolated relaxatio n of the trabecular meshwork (TM). This work was performed to investigate t he existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GT Pase)mediated pathway. METHODS. Isometric tension measurements of bovine TM and ciliary muscle (Ch l) were performed. Intra- and extracellular calcium buffering was accomplis hed with EGTA and 1,2-bis(2-aminophenoxy)-ethane-N,N,N,N',N'-tetra-acetic a cid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4 alpha -phorbol. Calcium-independent contracti on was blocked using the highly specific ROCK inhibitor Y-27632. Western bl ot analysis and immunoprecipitation was performed using human TM cells. RESULTS. In TM, carbachol induced partial contraction under conditions of e xtracellular calcium depletion (22.1% +/- 2.3% versus 100%, n = 9). The mem brane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%,, la = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 3) but not i n CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4 alpha -phorbol did not induce contraction, indicating that activation of PKC is i nvolved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blo t analysis and immunoprecipitation revealed the expression of the rho-A pro tein in human TM cells. CONCLUSIONS. The data indicate that contrary to CM, the TM features calcium -independent contractile mechanisms linked to rho-A and PKC isoforms that d o not require calcium for activation. ROCK inhibitors may allow specific mo dulation of the TM to enhance outflow facility, thus lowering intraocular p ressure.