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Abstract #15769 Published in IGR 2-3
Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A
Thieme H; Nuskovski M; Nass JU; Pleyer U; Strauss O; Wiederholt MInvestigative Ophthalmology and Visual Science 2000; 41: 4240-4246
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 4α-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 the 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 4α-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 the TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein in human TM cells. CONCLUSIONS: These 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.
Dr. H. Thieme, Institüt für Klinische Physiologie, Freie Universität Berlin, Germany
Classification:
11.14 Investigational drugs; pharmacological experiments (Part of: 11 Medical treatment)
