Hitoshi Shichi

Microsomal electron transfer system of bovine retinal pigment epithelium

Pigment epithelium microsomes are capable of catalyzing oxidation of NADH and NADPH in the absence of l -ascorbate. Ascorbate increases the oxidase activity, and the ascorbate-stimulated fraction of the activity is cyanide insensitive. Reduction of epithelium microsomes by the pyridine nucleotides under anaerobic conditions reveals the presence of a hemoprotein, which has absorption maxima at 429, 528 and 559 m μ in the (reduced vs. oxidized) difference spectrum. 3,4-Dihydroxyphenylalanine, which stimulates the pyridine nucleotide oxidation, is also capable of reducing the hemoprotein. The reduced form is rapidly autoxidizable and combines with carbon monoxide. From the carbon monoxide spectrum the microsome-bound hemoprotein was identified with P-450, a CO-binding hemoprotein originally found in liver microsomes. The hemoprotein could be solubilized by trypsinolysis of microsomes, purified on Sephadex and identified with P-420, a solubilized form of P-450. The spectral characteristics and amino acid composition of the solubilized component agree well with those of P-420 that we previously obtained from heart microsomes. Cytochrome b 5 , if present, must be a very minor hemoprotein component in pigment epithelium microsomes.

Patterns of Experimental Allergic Uveitis Induced by Rhodopsin and Retinal Rod Outer Segments

We have observed that both washed outer segments and purified rhodopsin produce primarily a posterior uveitis with little or no anterior segment inflammation if an effort is made to remove all contaminating soluble retinal antigen. The differences in the immunopathologic responses to rhodopsin compared to soluble retinal antigen may be explained by the solubility difference between these antigens. With the soluble retinal antigen there may be anterior segment inflammation and diffuse tissue necrosis. The allergic response to rhodopsin appears to be confined to the posterior segment with tissue necrosis in severe cases restricted to the anatomic distribution of rhodopsin in the outer retina.

GTP binding protein: properties and lack of activation by phosphorylated rhodopsin.

Taking advantage of the capability of GTP binding protein to bind GTP, we purified the catalytic subunit (G alpha) of bovine rod GTP binding protein by nucleotide-affinity chromatography on Blue Sepharose CL6B. Purified G alpha was essentially free of bound guanine nucleotide and activated by photoactivated rod membranes. Circular dichroism spectra suggested that a significant portion of the protein would be in alpha-helical conformation. No appreciable differences were detected in the circular dichroism spectra when G alpha . GDP and G alpha . GppNp were compared. The extent of G protein activation by rod membranes was reduced moderately by phosphorylation of rhodopsin during photolysis. However, if the pigment had been phosphorylated and regenerated, the ability of rhodopsin to activate G protein was markedly suppressed.

Tetranactin, a macrotetrolide antibiotic, suppresses in vitro proliferation of human lymphocytes and generation of cytotoxicity

Tetranactin, a hydrophobic cyclic antibiotic produced by Streptomyces aureus, has previously been shown to suppress in vitro activation of rat lymphocytes by concanavalin A as well as the onset of experimental autoimmune uveoretinitis in Lewis rats. Here we report the effects of tetranactin on human T and NK lymphocytes in vitro. Tetranactin, at concentrations up to 100 ng/ml, was not toxic to human lymphocytes but completely abrogated the proliferation of human T lymphocytes in response to allogeneic cells in mixed lymphocyte cultures. Tetranactin also blocked the initiation of proliferation in response to interleukin-2, but did not block proliferation of interleukin-2-activated cells. Tetranactin also blocked generation of cytotoxic T lymphocytes and activated killer cells in the mixed lymphocyte culture. However, up to 100 ng/ml tetranactin did not alter the lytic activity of cytotoxic T or NK lymphocytes generated in its absence. The ability of low doses of tetranactin to block the induction of lymphoproliferation is similar to the action of cyclosporin A. Since cyclosporin A is also a cyclic hydrophobic molecule, the immunosuppressive actions of these two agents may involve a similar mechanism.

Bovine non-pigmented and pigmented ciliary epithelial cells in culture: Comparison of catalase, superoxide dismutase and glutathione peroxidase activities

Non-pigmented epithelial (NPE) cells and pigmented epithelial (PE) cells were dissociated from bovine ciliary processes by brief digestion with pronase and grown in a culture medium containing high fetal bovine serum for at least 25 generations. Both types of cells grown to confluence showed the presence of intermediate junctions with associated tonofilaments. PE cells were distinguished from NPE cells by pigmentation during the early passages. Gamma-glutamyl transpeptidase activity was associated almost exclusively with NPE cells and proved to be a useful enzymatic marker to distinguish NPE from PE. A comparison was made between NPE and PE cells as to the levels of enzymes involved in the detoxification of active oxygen species. Catalase, Se-dependent glutathione peroxidase and superoxide dismutase activities were significantly higher in NPE than in PE cells. The results suggest that NPE cells play the major role in detoxification of active oxygen species during aqueous humor formation.

Superoxide in ocular inflammation: Human and experimental uveitis

A possible involvement of superoxide in the pathogenesis of uveal inflammation in man and experimental animals was investigated. Superoxide production by the leukocytes of Behcet patients was significantly higher in the attack phase than in the remission phase. Leukocyte superoxide generation was also enhanced in guinea pigs with S-antigen-induced experimental autoimmune uveoretinitis (EAU). If the animals were treated with superoxide dismutase (SOD) at the onset of EAU, aqueous humor cell count was significantly lower than that of control (i.e., without SOD treatment). Infiltration of the inflammatory cells in the anterior retina was markedly reduced in SOD-treated animals. A similar protective effect of SOD against tissue damage was also observed in a bovine serum albumin-induced passive Arthus type uveitis in rabbits. These results suggest that superoxide may play a role in causing tissue damage in animal models of ocular inflammation and possibly in Behcet disease.

Immunocytochemical study of phenobarbital-and 3-methylcholanthrene-inducible cytochrome P450 isozymes in primary cultures of porcine ciliary epithelium

We found in the previous study that the induction of 7-pentoxyresorufin O-dealkylase and 7-ethoxyresorufin O-dealkylase activities by phenobarbital and 3-methylcholanthrene, respectively, is more pronounced in porcine ciliary nonpigmented epithelial cells than in pigmented epithelial cells. In order to determine whether cytochrome P450 isozymes that mediate the O-dealkylase activities are also induced in nonpigmented cells under the conditions, primary cultures of porcine ciliary processes were treated with phenobarbital and 3-methylcholanthrene and the expression and localization of cytochrome P450 isozymes induced by these compounds were investigated by immunocytochemical methods using antibodies against the individual P450 isozymes. Intense labeling of nonpigmented epithelial cells was observed when ciliary processes treated with phenobarbital were reacted with anti-P450 (phenobarbital) antibody and when the processes treated with 3-methylcholanthrene were incubated with anti-P450 (methylcholanthrene) antibody. The labeling patterns supported the conclusion that the O-dealkylase activities and P450 isozymes specific for these activities are co-induced in and localized to the endoplasmic reticulum. This study is the first report presenting direct evidence of cytochrome P450 induction in primary cultures of ocular tissues and demonstrates the usefulness of porcine ciliary epithelial cells for studying the induction of ocular drug metabolizing enzymes.

[38] Rhodopsin kinase

Publisher Summary This chapter focuses on rhodopsin kinase. Rhodopsin kinase—the enzyme involved in the reaction—catalyzes the transfer of the terminal (γ) phosphate group of adenosine triphosphate (ATP) to the opsin protein. In the assay method of rhodopsin kinase, bovine rod outer segments are treated with urea to prepare the substrate. This treatment denatures membrane-associated rhodopsin kinase but has little effect on rhodopsin. The substrate is then incubated in the light with rhodopsin kinase, [y -32p ]ATP, and [adenine- 3 H]ATP. The inclusion of [ 3 H]ATP is to estimate the extent of binding of ATP itself. In an alternative method, rod membranes are incubated with [γ -32P ]ATP in dark and then in light. The kinase activity is determined by subtracting the 32 P incorporated in dark from the 32 P incorporated in light. The phosphorylated protein is identified as rhodopsin after the separation of the labeled protein by chromatographic and electrophoretic methods.

Purification and properties of glutahtione reductases from bovine ciliary body

Two glutathione reductases, designated GR-I and GR-II, were purified from bovine ciliary body to apparent homogeneity by ion-exchange chromatography on DEAE-agarose and gel filtration on Sephacryl S-200. The two enzymes were isolated whether protease inhibitors were included or not in the medium during tissue homogenization. GR-I was found to have a molecular weight of about 140,000 by gel filtration and be composed of two identical subunits. On the other hand, GR-II was shown to exist as aggregates with molecular weights larger than 670,000. The subunit molecular weight of GR-II was estimated to be about 45,000 by SDS-polyacrylamide gel electrophoresis. The high molecular aggregate dissociated into smaller species by treatment with mercaptoethanol. The isoelectric points of GR-I and GR-II were determined to be 5.8 and 6.4, respectively. The Km values for NADPH and oxidized glutathione (GSSG) were 22- and 84 microM for GR-I and 11- and 98 microM for GR-II. Coenzyme A-glutathione disulfide served as a substrate, though it was much less reactive than oxidized glutathione, for both enzymes. S-alkyl glutathiones were inactive as substrates. Nitrofurantoin was a non-competitive inhibitor of the enzymes. 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) inhibited GR-I and GR-II irreversibly. The two enzymes are similar, if not identical, in their amino-acid composition. The two enzymes are also very similar on the basis of the relative position of tryptic peptides separated in two-dimensional peptide maps. From the tissue content and turnover numbers of these enzymes it is concluded that bovine ciliary body has a sufficient capability for rapid reduction of oxidized glutathione produced in the tissue during glutathione-dependent peroxide detoxification.

Immunolocalization of prostanoid EP receptor isotypes in human trabecular meshwork.

Purpose. To assess the localization of the EP-type prostanoid receptors in the human trabecular meshwork (TM) and to determine their spatial distribution in relation to the contractile α-smooth muscle actin fibres. Methods. Cryosections of human anterior segments were obtained from 17 different donors and immunostained with different EP receptor subtype specific antibodies. Double staining for the EP2 receptor and smooth muscle actin was carried out. Western blots of TM protein samples were studied. Results. No specific staining for the EP1 receptor was observed. The antibodies against the EP2 receptor revealed in all donors intense staining of human trabecular cells throughout the meshwork. EP3 receptor specific staining was not detected. EP4 immunostaining was confined to the corneo-scleral region near Schwalbes line. On western blots, the EP2 receptor was detected. In the posterior TM, the EP2 receptor staining was associated with the dense network of actin fibres. Conclusions. These immunocytochemical results present evidence that the EP2 receptor is the most abundantly expressed isotype of the PGE receptors in the human TM. This conclusion is in agreement with our previous findings at the transcript level. The relaxant responses of the TM to application of EP2 receptor agonists, and flow enhancement evoked by prostaglandin PGE1, may be explained by the close spatial association of the EP2 receptor with actin fibres.