H-7 Disrupts the Actin Cytoskeleton and Increases Outflow Facility

Abstract

To determine the effects of the serinethreonine kinase inhibitor H-7 on (1) cell junctions and the attached actin-based cytoskeleton in cultured bovine aortic endothelial cells, and (2) outflow facility in living monkeys.Methods: Bovine aortic endothelial cells were cultured by standard techniques. The architecture and distribution of actin filaments, vinculin, and ␤-catenin in bovine aortic endothelial cells were studied by immunolabeling before and after exposure to H-7 at various concentrations and durations. Outflow facility (perfusion) and intraocular pressure (Goldmann tonometer) were determined before and after the intracameral or topical administration of H-7 or a vehicle.Results: In bovine aortic endothelial cells, exposure to H-7 produced a reversible time-and concentrationdependent disruption of actin microfilaments and an alteration in the organization of cell-cell and cell-matrix adhesions. In monkeys, intracameral and topical administration of H-7 dose dependently and reversibly doubled facility, and topical H-7 reduced intraocular pressures.Conclusion: H-7 increases outflow facility in monkeys, probably by inhibiting cell contractility, cytoskeletal support, and cell-cell adhesions in the trabecular meshwork.

Enhancing trabecular outflow by disrupting the actin cytoskeleton, increasing uveoscleral outflow with prostaglandins, and understanding the pathophysiology of presbyopia

“…Intracameral exchange with either of these compounds results in a dramatic several fold increase in outflow facility in the nonhuman primate eye after an initial delay. Similarly topical H-7 produced about the same doubling of outflow facility (Tian et al, 1998;.…”

“…Stimulating the Rho pathway and enhancing phosphorylation of the myosin light chain increases contractility. Conversely, inhibiting the Rho pathway and myosin light chain kinase with a variety of small molecules (H-7, ML-7, Y-27632) (Epstein et al, 1999;Rao et al, 2001;Tian et al, 1998) or with bacterial toxins such as C3 (Liu et al, 2005), will uncouple actin from myosin, resulting in relaxation of the cells and disassembly of the actin cytoskeleton. In addition to small molecules and toxins, there are other proteins, such as caldesmon, that uncouple the linkage of actin and myosin II resulting in focal adhesion disassembly and loss of actomyosin contractility (Helfman et al, 1999).…”

Extracellular matrix in the trabecular meshwork

“…(Bárány 1953;Van Buskirk and Brant 1974;Epstein et al 1981;Epstein and Anderson 1984;Suzuki and Anderson 1993) A number of agents that primarily act on cytoskeletal organization have been shown to modulate outflow facility. (Kaufman and Erickson 1982;Johnson 1997;Peterson et al 1997;Tian et al 1998;Epstein et al 1999;Sabanay et al 2000;Rao et al 2001;Vittitow et al 2002;Rao et al 2005) These studies suggest that cells somewhere in the TM or the SC inner wall, or both, are directly involved in forming the outflow resistance. Perfusion of a fibronectin fragment that disrupts integrin-ECM interactions and could affect either ECM or cytoskeleton also increases outflow facility.…”

Effect of H-7 on Secondary Cataract After Phacoemulsification in the Live Rabbit Eye

“…To prevent the possible inhibitory effect of IOLs on LEC migration from complicating the experimental results, IOLs were not implanted following phacoemulsification in this In previous studies, 300 mM H-7 maximally inhibits the actomyosin-driven contractility and perturbs the dynamics of the actin cytoskeleton and associated proteins in various cultured cells. [14][15][16] Because 1,200 mM intravitreal H-7 has shown a similar effect on the pupil's response to pilocarpine as 300 mM intracameral H-7 in monkeys, 26 the peak concentration of H-7 in the AC following each treatment of the 1,200 mM intravitreal H-7 could be *300 mM in the rabbit eye. Additionally, the effective PCO-inhibition concentration of H-7 in the cultured human lens capsule is also 300 mM.…”

“…H-7 (1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine), which inhibits several protein kinases including myosin light chain kinase (MLCK), Rho kinase, and protein kinases A and C, dramatically reduces actomyosindriven contractility, leading to cellular relaxation, deterioration of the microfilaments and perturbation of their membrane anchorage, and loss of stress fibers and focal contacts in various types of cultured cells. [14][15][16] Probably through these cytoskeletal modulations and related mechanisms, H-7 inhibits proliferation, migration, and EMT of several nontumor and tumor cell lines, [17][18][19] affects collagenase production and matrix metalloproteinase release in cultured human keratinocytes and airway smooth muscle cells, 20,21 and blocks wound healing in organ-cultured rat corneas. 22 Based on these findings, we hypothesize that H-7 might reduce the incidence and/or severity of secondary cataract after lens surgery.…”