BACKGROUND: Endothelin-1 (ET-1) is known to induce contraction of trabecular meshwork (TM) and is probably involved in the pathogenesis of glaucoma. Calcium (Ca(2+))-independent contraction has been shown in TM, and its inhibition may represent an interesting way of influencing outflow facility, and thus intraocular pressure (IOP). This study investigates the role of ET-1 and its receptors ET-A and ET-B (ET-AR and ET-BR) in TM Ca(2+)-independent contractility. METHODS: Isometric tension measurements of bovine TM (BTM) strips were performed using a force-length transducer system. Intra- and extracellular Ca(2+) buffering was achieved by means of EGTA and BAPTA-AM. Under Ca(2+)-free conditions, ET-1-induced contractility of TM was assessed also in the presence of the specific inhibitors for ET-AR and ET-BR, BQ123 and BQ788 respectively. In order to clarify the intracellular mediators of Ca(2+)-independent contractility induced by ET-1, TM contraction was further measured in the presence of Y-27632, a selective inhibitor of Rho-associated kinases (ROCKs). The expression of ROCK1 and of its activating protein RhoA in BTM cells was investigated using western blot analysis. RESULTS: ET-1 induced a significant contraction of native BTM after intra- and extracellular Ca(2+)-depletion (45% +/- 8% of the maximally inducible contraction). Both endothelin receptor inhibitors BQ123 and BQ788 significantly reduced TM Ca(2+)-independent contraction in response to ET-1 (8.4 +/- 3.3% and 20.3 +/- 4.8% respectively). In the presence of the ROCK inhibitor Y-27632, ET-1-induced contraction of TM under Ca(2+)-free conditions was almost completely abolished (4.3% +/- 1.7%, p < 0.001). A clear signal for RhoA at 24 kDa and ROCK1 at 160 kDa could be detected in lysates of native tissue and cultured BTM cells with western blot. CONCLUSIONS: This study shows evidence that a significant portion of ET-1-induced contraction of TM is Ca(2+)-independent. In this contraction pathway, both ET-AR and ET-BR are involved with RhoA and its kinases as intracellular mediators. Ca(2+)-independent contraction of TM in response to ET-1 may represent a specific target to modulate IOP.
BACKGROUND:Endothelin-1 (ET-1) is known to induce contraction of trabecular meshwork (TM) and is probably involved in the pathogenesis of glaucoma. Calcium (Ca(2+))-independent contraction has been shown in TM, and its inhibition may represent an interesting way of influencing outflow facility, and thus intraocular pressure (IOP). This study investigates the role of ET-1 and its receptors ET-A and ET-B (ET-AR and ET-BR) in TM Ca(2+)-independent contractility. METHODS: Isometric tension measurements of bovine TM (BTM) strips were performed using a force-length transducer system. Intra- and extracellular Ca(2+) buffering was achieved by means of EGTA and BAPTA-AM. Under Ca(2+)-free conditions, ET-1-induced contractility of TM was assessed also in the presence of the specific inhibitors for ET-AR and ET-BR, BQ123 and BQ788 respectively. In order to clarify the intracellular mediators of Ca(2+)-independent contractility induced by ET-1, TM contraction was further measured in the presence of Y-27632, a selective inhibitor of Rho-associated kinases (ROCKs). The expression of ROCK1 and of its activating protein RhoA in BTM cells was investigated using western blot analysis. RESULTS:ET-1 induced a significant contraction of native BTM after intra- and extracellular Ca(2+)-depletion (45% +/- 8% of the maximally inducible contraction). Both endothelin receptor inhibitors BQ123 and BQ788 significantly reduced TM Ca(2+)-independent contraction in response to ET-1 (8.4 +/- 3.3% and 20.3 +/- 4.8% respectively). In the presence of the ROCK inhibitor Y-27632, ET-1-induced contraction of TM under Ca(2+)-free conditions was almost completely abolished (4.3% +/- 1.7%, p < 0.001). A clear signal for RhoA at 24 kDa and ROCK1 at 160 kDa could be detected in lysates of native tissue and cultured BTM cells with western blot. CONCLUSIONS: This study shows evidence that a significant portion of ET-1-induced contraction of TM is Ca(2+)-independent. In this contraction pathway, both ET-AR and ET-BR are involved with RhoA and its kinases as intracellular mediators. Ca(2+)-independent contraction of TM in response to ET-1 may represent a specific target to modulate IOP.
Authors: Rita Rosenthal; Lars Choritz; Sebastian Schlott; Nikolaos E Bechrakis; Jan Jaroszewski; Michael Wiederholt; Hagen Thieme Journal: Exp Eye Res Date: 2005-06 Impact factor: 3.467
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