Motokazu Itoi

Collagenase in the cornea

A collagenase that can digest both collagen fibers in the stroma and the native collagen molecule was found in rabbit corneal epithelium, using a tissue culture assay. Calf, chicken and fetal calf serum added to the tissue culture media inhibited the collagenase, while human and rabbit serum were not inhibitory. Edathamil calcium disodium demonstrated a potent inhibitory effect on the enzyme with minimal toxicity to the epithelial cells. The clinical implications of a corneal collagenase are discussed.

Spreading of cultured corneal epithelial cells on fibronectin and other extracellular matrices.

Previous investigations demonstrated that fibronectin is an essential adhesive glycoprotein for the corneal epithelial cells. Fibronectin mediates attachment and spreading of the corneal epithelial cells. However, it seems to be important to know the changes in cellular receptor activity in order to understand the interactions of corneal epithelial cells and underlying extracellular matrix. In this investigation, we studied the effects of various culture times and conditions on the receptor activity of rabbit corneal epithelial cells for fibronectin. The cultured cells were placed on tissue culture plates that were coated with various concentrations of four extracellular matrices: fibronectin, laminin and collagen types I and IV. Freshly isolated corneal epithelial cells without culture did not spread on fibronectin and laminin; they spread only on collagen types I and IV. When the epithelial cells were cultured for 12 h or more, they spread on fibronectin. However, the spreading of the cells on collagen types I and IV was the same regardless of the culture period (up to 20 h). Only a small number of epithelial cells spread on laminin at the highest concentration examined after culture for 12 h or more. Thus, the corneal epithelial cells responded differently to fibronectin, collagen types I and IV, and laminin. Perhaps the receptor for fibronectin appears when epithelial cells are cultured, but the receptor(s) for collagen types I and IV are always present on the cell surface of corneal epithelial cells.

Specular Microscopic Studies of the Corneal Endothelia of Japanese Diabetics

We did a specular microscopic study with computer-assisted morphometric analysis of individual cells on the corneal endothelia of 69 Japanese diabetics and 53 age-matched nondiabetics. No statistically significant differences were seen in the mean cell areas of diabetics and nondiabetics. However, the coefficient of variation of cell area was significantly higher and the percentage of hexagonal cells was significantly lower in diabetics than in nondiabetics. Although racial differences of the corneal endothelia exist between Japanese and Americans, diabetic changes are quite similar in the two populations.

Glutathione isopropyl ester (YM737) inhibits the progression of x-ray-induced cataract in rats

The effect of glutathione (GSH) isopropyl ester on the progression of X-ray-induced cataract was investigated in rats. Intraperitoneal administration of 20 mg/kg GSH isopropyl ester, three times weekly, 1 day after a single irradiation dose delayed the progression of X-ray-induced cataracts significantly. The amount of non-protein SH groups and the Na+/K+ ratio in the lenses of drug-treated rats were maintained at the normal levels even 27 weeks after irradiation. Posttreatment with the drug resulted in a significantly lower level of malondialdehyde in the irradiated lenses than in the nontreated lenses. When 500 mg/kg GSH-isopropyl ester was administered by i.p. injection to normal rats, the GSH-ester was detected in plasma and aqueous humor after 15 min. In the lenses of the GSH-isopropyl ester-injected rats, the GSH level was 120% of that in the non-treated rats after 4 h, suggesting that GSH-isopropyl ester is transported from the aqueous humor to the lens and there converted to GSH after about 4 h. Our observations lead us to conclude that the delay of X-ray-induced lens opacity progression is due to maintenance of normal lenticular GSH levels achieved by post-irradiation administration of GSH-isopropyl ester. However, continuous administration of 100 mg/kg after irradiation had no effect on the progression of cataracts induced by X-rays.

Changes of glutathione and taurine concentrations in lenses of rat eyes induced by galactose-cataract formation or ageing

Sulphur-containing compounds in the lens were studied in relation to galactose cataract formation. Female Wistar rats were fed a 35% galactose diet and the changes in lens sulphur concentration and its distribution on a gel filtration column were compared with age-related changes. Concentration of sulphur in the whole lens decreased with time. A low constant level was attained on the fifth day of the galactose diet. A decrease of sulphur concentration in the soluble fraction of the lens paralleled that of the whole lens which was correlated with the decrease of glutathione and taurine concentrations on a gel filtration column by high-performance liquid chromatography-inductively coupled argon plasma atomic emission spectrometry (HPLC-ICP). Concentration of magnesium in the lens decreased after the fifth day, while the ratio of sodium to potassium increased. These changes in sulphur-containing compounds and metals were observed prior to the onset of cataract formation.

Visco-elastic properties of the lens

The dynamic visco-elastic properties of human, monkey and rabbit lenses have been measured with a dynamic rheometer. The value for the apparent elastic modulus (Young modulus) of the lens was found to be 10 5 –10 6 dyne/cm 2 in humans and 10 4 –10 5 dyne/cm 2 in monkeys and rabbits. The value for the loss tangent was 0·3–0·4 in the human lens and 0·3–0·6 in rabbit and monkey lenses. Elastic moduli and loss tangents of the lenses showed poor dependence on temperature at 15°–55°C and on frequency of oscillation at 0·01–25 c/s. The lenses showed linear viscoelasticity when the amplitude of oscillation was below 0·02 mm, and they showed non-linear visco-elasticity when the amplitude exceeded 0·03 mm.

Effects of 2-Mercaptopropionylglycine on the Development of X-Ray-Induced Cataract in Rats

The effect of 2-mercaptopropionylglycine on the development of cataract induced by a single dose of X-ray (10 Gy) was investigated in rats. Intraperitoneal injection of 20 mg/kg, three times weekly starting 1 day after irradiation delayed the development of X-ray-induced cataracts significantly. The amounts of non-protein SH groups, malondialdehyde and the Na+/K+ ratio, in the lenses of rats post-treated with the drug were significantly maintained at normal levels even at 27 weeks after irradiation. On the other hand, a single administration of 250 mg/kg of the compound, 30 min prior to irradiation had no effect on cataract progression induced by X-ray.

Peptidase in the cornea

Peptidase activity, operative at neutral pH, was found in bovine cornea and conjunctiva with a synthetic substrate assay. The same peptidase was found previously in commercial bacterial collagenase preparations. Cell fractionation of homogenates revealed the activity to be maximal in the high-speed supernatant of the corneal and conjunctival epithelium, and in the intermediate fraction of the endothelium. There was no peptidase found in the stroma. EDTA, cysteine, calf serum and calf aqueous inhibited this activity. The specificity and advantages of the substrate are discussed, and the clinical implications of a collagenase-like peptidase system within the cornea are noted.