Yukuo Yoshida

Reduction of intraocular pressure by topical administration of an inhibitor of the Rho-associated protein kinase

Purpose. Rho-associated coiled coil-forming protein kinase (ROCK) is a downstream target of a small GTPase, Rho. The kinase has been reported to regulate actomyosin-based contractility of smooth muscles by modulation of myosin phosphatase activity. Contractility of ciliary muscle could be implicated in regulation of intraocular pressure while that of ocular vessels could affect blood flow to retina. The present study has been performed to investigate the effect of a ROCK inhibitor on intraocular pressure in rabbits. Methods. An inhibitor of ROCK, Y-27632, was dissolved in an ophthalmic solution and topically administered to the eye of a Japanese white rabbit. Intraocular pressure was measured by pneumatonography (n = 12, 24 eyes). Constriction of ciliary muscle was measured by the Magnus method using 12 eyes. Results. Topical application of 0.1 and 0.03% Y-27632 significantly decreased the intraocular pressure, with maximum decreases of 5.3 and 4.3 mm Hg after 90 minutes compared with the control eye. Y-27632 inhibited the carbachol-induced constriction of rabbit ciliary muscle. Conclusions. The ROCK inhibitor reduced intraocular pressure in rabbits by topical instillation. The inhibitor relaxed the excised ciliary muscle which was previously constricted by carbachol suggesting that the inhibitor acts to increase the uveoscleral outflow. Our results suggest that the ROCK inhibitor is a promising treatment for glaucoma therapy in the next generation.

Involvement of calpain in hypoxia-induced damage in rat retina in vitro.

Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N2/5% CO2. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate alpha-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of mu- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa alpha-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro.