Calpain II, a high Ca2+‐requiring form of Ca2+‐dependent cysteine proteinase (EC 3.4.22.17), isolated from bovine lens was found to cleave actin and vimentin, two major cytoskeletal elements of the lens. Polyacrylamide gel electrophoresis revealed that actin (M r 43000) was broken down through intermediary products of approximate M r 42000 and 40000, while vimentin (M r 57000) was rapidly cleaved into several fragments ranging from M r 44000 to 20000. The cleavage was dependent on Ca2+ and could be blocked by calpastatin, a calpain‐specific inhibitor. These findings suggest that calpain might play a role in age‐related degradation of the lens cytoskeleton.

Cultured chick retinal pigment epithelial cells phagocytosed polystyrene latex particles. The phagocytosis was inhibited very specifically by melatonin, which attained 50% inhibition at about 10^––16M. Other indole compounds such as 5-methoxytryptophol, 5-hydroxytryptophol, 6-hydroxymelatonin, N-acetylserotonin, 5-methoxytryptamine and serotonin were also inhibitory although their effects were less than 1/10,000 that of melatonin. Possible retinal neurotransmitters, acetylcholine, γ-aminobutyric acid, glycine, dopamine, aspartic acid and glutamic acid, had no or only a minimum inhibitory effect, and was also the case for prostaglandin D₂, E₂, F₂α, and I₂. Taurine was not inhibitory at all. Among nucleotides, cyclic AMP specifically inhibited phagocytosis, giving 50% inhibition at about 10^––11M. Melatonin inhibition was increased by copresence of isobutylmethylxanthine. Inhibition by either melatonin or cyclic AMP was reversed by dibutyryl cyclic GMP. The reversal was observed also with compounds which were expected to increase intracellular cyclic GMP. Prostaglandin D₂ reversed inhibition in both cases, but its effect was incomplete and per se it had an inhibitory effect. Melatonin derivatives reversed inhibition by melatonin alone but not inhibition by cyclic AMP. Taurine efficiently reversed both kinds of inhibition. Other possible neurotransmitters were ineffective. Taurine was thus the most effective of these compounds. We suggest the following hypothetic control mechanism of phagocytic activity of the pigment epithelial cells: melatonin and cyclic AMP are intercellular and intracellular signals, respectively, of stopping phagocytosis, while taurine and cyclic GMP are intercellular and intracellular signals, respectively of cancelling this stop signal. Phagocytic activity of chick retinal pigment epithelial cells might be regulated by the concentration ratio of melatonin to taurine in the interphotoreceptor space.

Utilizing horseradish peroxidase as a tracer, electron microscopic studies were done on the blood-optic nerve and fluid-optic nerve barrier to the peroxidase diffusion. Following intravenous injection the peroxidase was observed to fill the lumen of the capillaries of the laminar, prelaminar and orbital portions of the optic nerve but there was no penetratation of the capillary walls. The obstruction of the tracer diffusion out of capillary walls was attributed to the tight junctions between the endothelial cells. Peroxidase penetration was also absent in the capillaries of the pia and dura mater, however, was observed in pinocytotic vesicles of the endothelial cells. Lateral diffusion from the surrounding choroid into the optic nerve was detected but diffusion from the prelaminar optic nerve into the juxta-optic nerve retina was prevented by the Kuhnt intermediary tissue. Tight junctions which prevented peroxidase diffusion were found between the glial cells of the Kuhnt tissue, and this tissue was the barrier between the prelaminar optic nerve and the juxta-optic nerve retina. Peroxidase which was given into the lateral ventricle of the brain appeared in the subarachnoidal space around the optic nerve and penetrated freely into the optic nerve. The pial surface of the optic nerve possess no barrier activity. Peroxidase could be traced along the intercellular space between glial cells and optic nerve fibers. The basal lamina of the optic nerve capillaries was filled with peroxidase but diffusion into the capillary lumen was obstructured by the tight junctions between the endothelial cells.

No abstract

The age-related changes of calpain II (high-Ca2+-requiring form of Ca2+-dependent cysteine proteinase; EC 3.4.22.17) and alpha-crystallin in the lens of hereditary cataract (Nakano; cac/cac) mouse were studied. Before the onset of the cataract formation, i.e., at the end of the 2nd week after birth, the calpain activity in Nakano mice was as high as that in the control ICR mice, but it decreased rapidly as the cataract progressed to completion during the 4th and the 12th week. Marked degradation of lens proteins ensued between the 2nd and the 4th weeks, and one of these proteins was identified, using monospecific antibodies, as B chain of alpha-crystallin. A chain of alpha-crystallin was not degraded in vivo, in contrast to its known susceptibility to calpain in vitro. The present data suggest that in Nakano mice, calpain may be involved in the onset or early stage of the cataract formation.