Isamu Tsukahara

Phagocytic Activity of Cultured Retinal Pigment Epithelial Cells from Chick Embryo: Inhibition by Melatonin and Cyclic AMP, and Its Reversal by Taurine and Cyclic GMP

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(-16) M. 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, gamma-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, F2 alpha, and I2. Taurine was not inhibitory at all. Among nucleotides, cyclic AMP specifically inhibited phagocytosis, giving 50% inhibition at about 10(-11) M. 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.

Limited proteolysis of bovine lens α-crystallin by calpain, A Ca2+-dependent cysteine proteinase, isolated from the same tissue

A Ca2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) was found in the cystosolic fraction of bovine lens and purified to apparent homogeneity. The purified enzyme required 1 mM Ca2+ for its full activation and was composed of two subunits of Mr 80 000 and 29 000 as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). This enzyme, when activated by Ca2+, degraded both A- and B-chains of alpha-crystallin, which were isolated also from bovine lens. SDS-gel electrophoresis of the digest revealed that the A-chain (Mr 19 500) was broken down to produce an 18-kDa polypeptide fragment and the B-chain (Mr 22 500) to produce a 19.5-kDa polypeptide fragment. No further cleavage occurred even upon prolonged incubation or after the second addition of the enzyme, indicating the uniquely limited proteolysis of each chain protein. The existence of calpastatin, an endogenous inhibitor protein specific for calpain, was also demonstrated in bovine lens cytosol.

Degradation of actin and vimentin by calpain II, a Ca2+-dependent cysteine proteinase, in bovine lens

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.

An electron microscopic study on the blood-optic nerve and fluid-optic nerve barrier

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. Mit Hilfe von Meerrettich-Peroxidase wurden elektronenmikroskopische Tracer-Studien an der Blutsehnerven- und Liquorsehnervenbarriere unternommen. Nach intravenöser Injektion von Peroxidase füllte diese das Lumen der Capillaren des laminaren, prälaminären und Orbitalen Anteiles des Nervus opticus aus, ohne daß eine Penetration der Capillarwände zu beobachten war. Die Verhinderung einer Tracerdiffusion durch die Capillarwand wurde den “tight junctions” zwischen den Endothelzellen zugeschrieben. Auch bei den Capillaren der Pia und Dura fand sich keine Penetration von Peroxidase. Es wurden jedoch mit Peroxidase gefüllte pinocytotische Vesikel im Endothel beobachtet. Während eine laterale Diffusion von Peroxidase von der umgebenden Chorioidea in den Sehnerven festgestellt wurde, wurde eine Diffusion des Tracers vom prälaminären Anteil des Sehnerven in das umgebende Retinagewebe durch das Kuhntsche Zwischengewebe blockiert. “Tight junctions”, die eine Diffusion von Peroxidase verhinderten, fanden sich zwischen den Gliederzellen des Kuhntschen Gewehes, so daß dieses Gewebe als Barriere zwischen dem prälaminären Anteil des Sehnerven und der juxtapapillären Netzhaut wirkte. Peroxidase, die in einen Seitenventrikel des Gehirns appliziert wurde, erschien in dem den Sehnerven umgebenden Subarachnoidalraum und diffundierte ungehindert in den Sehnerven. Die Pia Mater des Sehnerven hatte somit keine Barrierenfunktion. Peroxidase konnte entlang den Intercellularspalten zwischen Gliazellen und Sehnervenfasern verfolgt werden. Die Basalmembran der Capillaren des Sehnerven war angefüllt mit Peroxidase. Eine Diffusion in das Capillarlumen wurde jedoch durch die “tight junctions” zwischen den Endothelzellen verhindert.

Kuhnt intermediary tissue as a barrier between the optic nerve and retina

Utilizing adult albino rats and lanthanum nitrate as a tracer, electron microscopic studies were done to provide additional information on the blood-optic nerve barrier. Following injection into the cervical artery, lanthanum was observed to fill the intercellular space of the optic nerve parenchyma. Diffusion from the prelaminar optic nerve into the juxta-optic nerve retina was, however, prevented by the Kuhnt tissue at the level of the rod and cone layer. In this region, tight junctions were found between the glial cells of the Kuhnt intermediary tissue and were continuous from the tight junctions of the retinal pigment epithelial cells to the junctions (zonulae adhaerentes) of the outer limiting membrane. From the level of the outer limiting membrane to the inner limiting membrane there was no diffusion of lanthanum into the adjacent retina despite the absence of tight junctions, and the lanthanum which had diffused from the choroid was never observed in the area of the inner limiting membrane of the optic nerve head. It is in this region that the functional barrier may exist.

Age-related changes of calpain II and α-crystallin in the lens of hereditary cataract (Nakano) mouse

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.

Acute severe uveitis with retinal vasculitis and retinal detachment.

5 adult patients (7 eyes) with acute severe uveitis with retinal vasculitis and retinal detachment of unknown etiology were studied. Yellowish exudations which appeared in the peripheral fundus rapidly extended posteriorly, and there was a patchy perivasculitis, white sheathing and obliteration of the retinal vessels. The absorption of these yellowish exudations was followed by atrophy of the retina and the choroid. Secondary detachment of the retina then developed, and the visual acuity remained poor in most cases.

Phagocytic activity of Cultured retinal pigment epithelium : uptake of polystyrene spheres and staphylococcus aureus

Abstract Phagocytosis of the chick embryo retinal pigment epithelial (RPE) cells in cultures was examined morphologically. All spheres such as native and glutaraldehyde-fixed Staphylococcus aureus ( S. aureus ) inoculated to the RPE cell cultures were actively phagocytized as well as polystyrene latex particles (PS). There was no significant difference in uptake rate between PS and fixed bacteria, although native bacteria were taken up at a somewhat higher level than the other spheres, at least up to 90 min after inoculation. Intracytoplasmic multiplication of native bacteria did not occur up to this early stage of inoculation, but after 20 hr, the delayed growth in the RPE cells was observed. In many phagosomes containing native bacteria, a characteristic membrane configuration which was apparently continuous with rough surfaced endoplasmic reticulum of the RPE cells was seen. On the contrary, these structures were never encountered in the phagosomes containing PS and fixed bacteria.

Experimental Chloroquine Retinopathy

Chloroquine retinopathy was produced experimentally in the eye of the albino corydoras (one of the tropical fish) by daily administration of chloroquine (0.1 mg per os). The enucleated eyes were examined from the 14th day to 3 months after the beginning of drug administration under light and electron microscopy. The first change of retina was the appearance of membraneous cytoplasmic body (MCB) in the cytoplasm of ganglion, amacrine, bipolar and horizontal cells. MCB might be degenerated lysosome. They showed lamellar figures or crystalline lattice-like structures. Secondarily, these MCB appeared in the inner segments of photoreceptor cells. The outer segments of rod cells disappeared, and then those of cone cells. Although photoreceptor cells were diminished in number in advanced degeneration, the cells of inner nuclear layer and ganglion cells were maintained in number. The presence of MCB dose not mean death of cells. The retinal pigment epithelial cells contained MCB in its cytoplasm only in severe degenerative cases, and did not show other remarkable changes. MCB also appeared in the cytoplasm of pericytes of retinal vessels. Chloroquine is considered to damage directly photoreceptor cells most severely.

Ultracytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the lacrimal gland of the rat.

SummaryThe electron-microscopic localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex was studied in the exorbital lacrimal gland of the untreated rat with the use of a newly developed one-step lead-citrate method (Mayahara and Ogawa 1980; Mayahara et al. 1980). In the rat lacrimal gland fixed for 15 min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, an electron-dense reaction product was observed on the plasma membrane of the basal infoldings and the lateral interdigitations of the ductal cells. The most intense reaction product — and thus the major site of the Na-K-ATPase activity — was evident on the basolateral membranes of the cells of the large interlobular ducts; a weak reaction was seen on the basolateral, extensively folded plasma membranes of the small intercalated ducts; no reaction product was observed on the plasma membranes of the acinar cells. Addition of 1) 10 mM ouabain, 2) p-chloromercuri-phenyl-sulfonic acid (PCMB-S), 3) elimination of K-ions from the incubation medium, or 4) preheating abolished completely the K-NPPase reaction. The activity was also substrate-dependent.Mg-ATPase-activity was observed not only in the basolateral membranes of all ductal cells but also in the basal part of the acinar cells and on the walls of blood vessels. This reaction was neither inhibited by ouabain nor activated by K-ions. The precipitate of the Mg-ATPase-activity was localized at the extracellular side of the plasma membrane, whereas the K-NPPase-reaction product was restricted to the cytoplasmic side of the plasmalemma.In contrast, non-specific alkaline-phosphatase (ALPase) activity was missing in cells of the large interlobular ducts, but obvious on the apical plasmalemma of cells lining the small intercalated ducts.With respect to its localization and reactivity pattern the activity of the K-NPPase (member of the Na-K-ATase complex) differs markedly from the Mg-ATPase- and ALPase-activity.