H Thieme1, M Nuskovski, J U Nass, U Pleyer, O Strauss, M Wiederholt. 1. Institut für Klinische Physiologie, and. Augenklinik Universitätsklinikum Benjamin Franklin, Freie Universität Berlin. Klinik und Poliklinik für Augenheilkunde, Charitè, Campus Virchow Klinikum, Humboldt Universität Berlin, German.
Abstract
PURPOSE: Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may represent a new way of influencing outflow facility through isolated relaxation of the trabecular meshwork (TM). This work was performed to investigate the existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GTPase)-mediated pathway. METHODS: Isometric tension measurements of bovine TM and ciliary muscle (CM) were performed. Intra- and extracellular calcium buffering was accomplished with EGTA and 1, 2-bis(2-aminophenoxy)-ethane-N,N:,N:,N:',N:'-tetra-acetic acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4alpha-phorbol. Calcium-independent contraction was blocked using the highly specific ROCK inhibitor Y-27632. Western blot analysis and immunoprecipitation was performed using human TM cells. RESULTS: In TM, carbachol induced partial contraction under conditions of extracellular calcium depletion (22. 1% +/- 2.3% versus 100%, n = 9). The membrane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%, n = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 9) but not in CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4alpha-phorbol did not induce contraction, indicating that activation of PKC is involved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein 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 do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.
PURPOSE: Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may represent a new way of influencing outflow facility through isolated relaxation of the trabecular meshwork (TM). This work was performed to investigate the existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GTPase)-mediated pathway. METHODS: Isometric tension measurements of bovine TM and ciliary muscle (CM) were performed. Intra- and extracellular calcium buffering was accomplished with EGTA and 1, 2-bis(2-aminophenoxy)-ethane-N,N:,N:,N:',N:'-tetra-acetic acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4alpha-phorbol. Calcium-independent contraction was blocked using the highly specific ROCK inhibitor Y-27632. Western blot analysis and immunoprecipitation was performed using human TM cells. RESULTS: In TM, carbachol induced partial contraction under conditions of extracellular calcium depletion (22. 1% +/- 2.3% versus 100%, n = 9). The membrane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%, n = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 9) but not in CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4alpha-phorbol did not induce contraction, indicating that activation of PKC is involved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein 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 do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.
Authors: Patricia K Russ; Asher I Kupperman; Sai-Han Presley; Frederick R Haselton; Min S Chang Journal: Invest Ophthalmol Vis Sci Date: 2009-07-23 Impact factor: 4.799