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M. Fernandez, D.L. Epstein, P.V. Rao; Characterization of the Cellular Mechanisms Involved in Forskolin–Induced Trabecular Meshwork Relaxation: Implications for Aqueous Humor Outflow Function . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1863.
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Agents that increase intracellular cAMP have been reported to increase aqueous humor outflow in various animal models. A mechanistic understanding of this effect, however, is lacking. The status of contractile and relaxation activity of trabecular meshwork has been suggested to regulate aqueous outflow. We therefore investigated the effects of forskolin, a well characterized inducer of cAMP production, on the relaxation status, actin cytoskeletal organization, and cell adhesions in cultured TM cells and on aqueous outflow in a perfusion model.
The dose response effects of forskolin on cell morphology, actomyosin organization, myosin II phosphorylation, and cell adhesions in primary porcine and human TM cells were determined by immunofluorescence and immunoblotting analysis. To determine the role of protein kinase A (PKA), protein kinase G (PKG), and Rho GTPase in forskolin–induced morphological and cytoskeletal changes, TM cells were treated with selective inhibitors of PKA (KT5720) or PKG (KT5823) prior to forskolin treatment. TM cells were also expressed with constitutively active Rho GTPase through an adenoviral vector. In addition, TM tissue isolated from forskolin–perfused porcine eyes was tested for the status of myosin II phosphorylation. These changes were compared to alterations in aqueous outflow facility.
Forskolin (1 to 10 µM) reversibly induced dramatic changes in TM cell shape (cell thinning, cell–cell detachment) and decreased actin stress fibers and focal adhesions. These changes were associated with a marked decrease in myosin II phosphorylation. Forskolin–induced cytoskeletal and morphological changes were completely prevented by constitutively active Rho GTPase. Furthermore, perfusion of forskolin (10 µM) in enucleated porcine eyes decreased TM myosin II phosphorylation in association with increased aqueous outflow facility within 5 hours of drug perfusion.
This data reveals an association between forskolin–induced increases in aqueous humor outflow facility, decreased myosin II phosphorylation, cell shape changes, actin depolymerization, and decreased cell adhesions. The study demonstrates a potential involvement of Rho GTPase in forskolin–induced TM relaxation and aqueous outflow regulation.
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