Immunocytochemical study of cytochrome P450 4A induction in mouse eye.
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Animals↗
Biomedical subjects
H Shichi
Publications and source records attributed to H Shichi.
Effect of prostaglandins on cultured porcine iris sphincter muscle cells.
Porcine iris sphincter muscle strips contracted in response to carbachol. The tissue contraction was inhibited by prostaglandin (PG) E2 but not by PGF2 alpha. In order to investigate the effect of PGs on the iris cells, the porcine sphincter muscle cells were grown in culture to a confluence and characterized. Using the secondary culture of cells, the effect of PGs on carbachol-induced cell contraction was investigated. Both PGE2 and PGF2 alpha at 100 microM blocked cell contraction completely. The concentration required to inhibit 50% of the maximum contraction in 15 minutes was 10(-6) M for PGE2 and 10(-6)-10(-7) M for PGF2 alpha. Using PGE2 receptor subtype agonists (EP2 agonist, 11-deoxy-16, 16 dimethyl PGE2 and EP3 agonist, sulprostone), PGE2 receptor involved in the inhibition of carbachol-induced contraction was identified to be of the EP2 subtype. In support of this characterization, the addition of PGE2 to cultured porcine sphincter muscle cells increased intracellular cAMP level. The discrepancy in PGF2 alpha effect on carbachol-induced sphincter muscle contraction between iris tissue strips and cultured cells suggests that nonmuscular cells may be involved in the modulation of the PGF2 alpha effect on sphincter muscle cells in vivo.
Animals↗
Induction of immunotolerance in rats by intratesticular administration of an eicosapeptide of bovine S-antigen.
Immunization of albino LEW rats with a retinal soluble antigen (S-antigen) induces experimental autoimmune uveoretinitis (EAU) which shows clinical features resembling those of human uveitis. Several uveitogenic epitopes have been identified in the antigen. This study reports that an intratesticular injection of low doses of a uveitogenic eicosapeptide (P343-362) of S-antigen prior to immunization with the same peptide prevented the onset of EAU by inducing systemic tolerance, designated orchidic tolerance. Splenic lymphocytes of both CD4+ and CD8+ subsets from tolerized rats transferred orchidic tolerance to syngeneic recipients and protected them from subsequent EAU induction. Orchidic tolerance elicited by low antigen dosage was mediated, in part, by active suppression due to suppressor or regulatory cells. At high antigen doses, however, regulatory activity was reduced possibly due to the induction of anergy in regulatory cells, and EAU severity increased. The CD4+ regulatory T cells from tolerized rats showed enhanced expression of IL-4 mRNA compared with CD4+ cells from control rats. Increased immunoreactivity for IL-4, IL-10 and TGF-beta was observed in the spleen and lymph nodes of tolerized animals. The results suggest that orchidic tolerance induced by low doses of P343-362 is mediated in part by CD4+ regulatory cells secreting Th2 cytokines.
Adoptive Transfer↗
Experimental autoimmune uveoretinitis induced by the gamma-subunit of cyclic guanosine monophosphate phosphodiesterase in rats.
PURPOSE: To investigate the capacity of the recombinant gamma-subunit (P gamma) of cyclic guanosine monophosphate phosphodiesterase to induce experimental autoimmune uveoretinitis in Lewis rats. METHODS: Bovine P gamma was expressed in Escherichia coli cells and purified by fast protein liquid chromatography. Lewis rats were immunized by a single footpad injection of P gamma emulsified in complete Freund's adjuvant. Clinical and histopathologic changes in the eye and pineal gland were examined. Lymphocytes were prepared from the lymph nodes of rats with uveitis and transferred by intraperitoneal injection to naive recipient rats. RESULTS: Immunization of rats with P gamma induced panuveitis and pinealitis with clinical and histopathologic changes similar to those induced by S-antigen. Lymphocytes from the lymph nodes of diseased rats transferred uveitis to naive recipients. CONCLUSIONS: P gamma, a retina-specific protein of molecular weight less than 10,000 kDa, is capable of inducing uveoretinitis in Lewis rats. The disease can be transferred adoptively to naive rats by injection of lymphocytes from donor rats with experimental autoimmune uveoretinitis. Inflammation of the pineal gland of immunized rats suggests that P gamma is not only localized to the retina but also to the pineal gland.
3',5'-Cyclic-GMP Phosphodiesterases↗
Tissue repair in the iris in experimental autoimmune uveoretinitis.
Our previous work on rats with S-antigen-induced experimental autoimmune uveoretinitis showed iris tissue changes involving infiltration of inflammatory cells, destruction of the iris architecture, and breakdown of the blood-retinal barrier. The present study reports the remarkable ability of the iris to regenerate during the postinflammatory period. The iris regenerates 50% of its architecture by Day 20 postimmunization. The number of lymphocytes and polymorphonuclear leukocyte (PMNs) was relatively high at this stage. Many capillaries showed abnormal endothelial cells. Unmyelinated nerve axons were often seen near blood vessels. The iris stroma was edematous. By Day 30, approximately 95% of the iris had regenerated, the number of lymphocytes and PMNs decreased, and the number of macrophages increased. Most capillaries looked normal and numerous axons in different stages of myelination were apparent. The stroma, the dilator muscle, and the posterior epithelium were almost completely restored. By Day 45, the iris appeared to be virtually normal. Most striking was the abundance of myelinated nerve axons located near blood vessels. Type I collagen immunoreactivity in vascular endothelial cells increased from Day 20 to Day 60 postimmunization, suggesting that blood vessel endothelial cells may play a role in collagenization of the iris stroma.
Animals↗
Histocytological study on the possible mechanism of acetaminophen cataractogenesis in mouse eye.
Ocular toxicity of acetaminophen was investigated in cytochrome P450 inducer-responsive and nonresponsive strains of mice by light and electron microscopy. Acetaminophen injected into C57BL6 mice (responsive strain) that had been pretreated with beta-naphthoflavone produced cataract. The drug did not induce cataract in C57BL6 mice without the pretreatment or in DBA2 mice (nonresponsive strain) similarly pretreated with beta-naphthoflavone. Therefore, induction of cytochrome P450 enzymes that metabolically activate acetaminophen is essential for cataractogenesis. Following acetaminophen injection, tissue damage became noticeable first in the ciliary epithelium and then spread to the iris, corneal endothelium, and lens. The neural retina, retinal pigmented epithelium, and choroid remained unaffected. A close examination of tissues revealed that mitochondria are the primary target of acetaminophen cytotoxicity in ocular tissues affected. The nucleus, endoplasmic reticulum, and other subcellular structures appeared normal. The course of propagation of tissue damage and the almost exclusive damage to mitochondria suggest that the cytotoxic metabolite of acetaminophen is secreted with the aqueous humor by the ciliary epithelium and transported to the lens and that inhibition of mitochondrial energy metabolism, together with other effects of the metabolite, contributes to acetaminophen-induced cataract.
Acetaminophen↗
Immunocytochemical study of cytochrome P450 (1A1/1A2) induction in murine ocular tissues.
C57BL/6 and DBA/2 mice were injected intraperitoneally with beta-naphthoflavone in corn oil and killed 48 hr later. Control animals received an injection of corn oil. The immunoreactivity of cytochrome P450 1A1/1A2 expressed in different ocular tissues and liver was examined with goat anti-P450 antibody (primary antibody) and gold-conjugated anti-goat antibody (secondary antibody). DBA/2 mice, which are non-responsive to aryl hydrocarbon treatment, showed negligible levels of immunoreactivity toward anti-P450 1A1/1A2 antibody in all ocular tissues, whether or not the animals were treated with beta-naphthoflavone. In responsive C57BL/6 mice, however, the immunoreactivity of the uveal tissues, especially ciliary non-pigmented epithelium, was markedly increased by beta-naphthoflavone treatment. The time course of induction of P450 1A1/1A2 immunoreactivity was very similar for the liver and ciliary non-pigmented epithelium, although the maximum level of immunoreactivity of the ciliary epithelium reached in 48 hr after inducer treatment was about 25% of that of liver. The present results support our previous observations that the P4501A enzyme activities (e.g. aryl hydrocarbon hydroxylase) in the liver and eye of C57BL/6 mice are under the same genetic regulation. Further, this study is the first demonstration of P450 isoform induction in specific ocular tissues of the whole animal.
Animals↗
Immunocytochemical localization of prostaglandin E2 receptor subtypes in porcine ocular tissues. I. Uveal tissues.
Polyclonal antibodies were raised against 15-residue sequences in the carboxyl terminal region of mouse EP1, EP2, and EP3 subtypes. The selected sequences are well conserved in different species. Using the antibodies, the localization of the receptor subtypes in porcine uveal tissues was investigated by immunoperoxidase reaction (by light microscopy) and immunogold labeling (by electron microscopy). EP1 immunoreactivity was found in ciliary nonpigmented epithelium and iris muscles (both sphincter and dilator). EP2 was localized to ciliary nonpigmented epithelium and muscle, iris sphincter muscle, and trabecular meshwork. EP3 immunoreactivity was detected in all uveal tissues examined.
Amino Acid Sequence↗
Immunocytochemical localization of prostaglandin E2 receptor subtypes in porcine ocular tissues. II. Nonuveal tissues.
The localization of PGE2 receptor subtypes (EP1, EP2 and EP3) in nonuveal tissues of porcine eye was investigated by immunocytochemical methods with polyclonal antibodies directed against conserved peptide sequences in the carboxyl terminal region of the receptor subtypes. EP1 was found only in corneal epithelium, while EP2 immunoreactivity was associated with corneal epithelium and endothelium and lens epithelium. EP3 receptor subtype was localized to all ocular tissues examined (except lens fiber cells), i.e. corneal epithelium and endothelium, lens epithelium, retinal photoreceptor and pigmented epithelium, Bipolar cell, horizontal cell, amacrine cell, ganglion cell and Muller cell.
Animals↗
The effects of prostaglandins E2 and F2 alpha on porcine ciliary muscle cells in culture.
The purpose of this work is to investigate the effect of prostaglandins (PGs) on the contraction of ciliary muscle cells. It has been proposed that PGs induce relaxation of ciliary muscle and facilitate uveoscleral outflow, and reduce intraocular pressure (IOP). The ocular response to PGs is complicated because the relative contributions of uveoscleral flow and the conventional outflow to lowering IOP and the type of PG receptors associated with ciliary muscle may vary depending on animal species. In order to obtain insights into prostaglandin receptors of ciliary muscle, ciliary muscle cells from porcine eye were grown in culture and characterized immunocytochemically with antibodies against smooth muscle-alpha-actin and PGE2 receptor subtypes. As in ciliary muscle tissues, positive immunostaining for alpha-actin and EP2 and EP3 subtypes was observed in cultured cells. Time-dependent contraction of cultured cells induced by 10(-4) M carbachol was recorded by taking sequential photographs and analyzed. Using this assay method, the effect of prostaglandins E2 and F2 alpha to inhibit the carbachol-induced contraction was studied. PGE2 showed potent inhibition of cell contraction; 10(-7) approximately 10(-8) M PGE2 inhibited 50% of full contraction in 15 min. PGF2 alpha at 10(-4) M neither caused cell contraction by itself nor blocked carbachol-induced contraction. The EP2 agonist 11-deoxy-16, 16-dimethyl PGE2 at 10(-4) M inhibited cell contraction but the EP3 agonist sulprostone had no effect. Dibutyryl cAMP at 3 x 10(-5) M inhibited contraction by 50%. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), less than 10(-7) M dibutyryl cAMP caused 50% inhibition. In support of the cAMP effect, the addition of 10(-4) M PGE2 to cultured cells in the presence of indomethacin and IBMX was shown to cause an 80% increase in intracellular cAMP concentration compared with the basal (i.e. unstimulated) level of cAMP. Stimulation of cells with 10(-4) M PGF2 alpha caused no increase in cellular cAMP. These results indicate that PGE2 receptor EP2 subtype, but not PGF2 alpha receptor, is involved in the inhibition (hence relaxation by inference) of carbachol-induced porcine ciliary muscle cell contraction. It awaits further studies to determine whether cultured ciliary muscle cells of other species respond similarly to different PGs.
1-Methyl-3-isobutylxanthine↗
Ultrastructural and immunocytochemical studies of smooth muscle cells in iris arterioles of rats with experimental autoimmune uveoretinitis.
In this study, we report on the ultrastructural and immunocytochemical changes that occur in smooth muscle cells of iris arterioles in S-antigen-induced experimental autoimmune uveoretinitis (EAU). The inflammatory phase (8-10 days postimmunization) was marked by infiltration of lymphocytes and polymorphonuclear leukocytes and monocytes in the iris stroma and perivascular tissue. Smooth muscle cells became hypertrophic with an 11.5-fold average increase in cell volume compared with control cells. In some of the cells, there was a marked increase in endoplasmic reticulum, ribosomes, and Golgi elements and a concomitant decrease in myofilaments, similar to that reported previously (Wang et al., Curr. Eye Res. 13, 747-754, 1994). However, the majority of hypertrophic smooth muscle cells showed only a slight increase in these synthetic organelles while retaining large amounts of myofilaments. There was no evidence for the migration or mitosis of the hypertrophic cells. Immunogold (IG) labeling of hypertrophic smooth muscle cells revealed changes in the immunoreactivity of several antigens. Labeling density for type I collagen increased progressively between 8 and 10 days, while that of decorin was slightly increased at 8 days and decreased at 10 days postimmunization. IG labeling for an alpha-actin isoform was significantly increased during the 8-10 day period, while that of beta-actin isoform was decreased. The results suggest that hypertrophic smooth muscle cells do not fully modulate to the kind of synthetic phenotype described in aortic smooth muscle cells. The significance of the transition in immunoreactivity from alpha- to beta-actin isoform is not known although it may reflect an increased synthetic state of muscle cells. The increased immunoreactivity of type I collagen and the changes in decorin, on the other hand, suggest that smooth muscle cells in EAU may be involved in remodeling of the extracellular matrix.
Actins↗
Phosrestin I, an arrestin homolog that undergoes light-induced phosphorylation in dipteran photoreceptors.
Two classes of phosphorylated homologs of vertebrate arrestins, designated phosrestins I (PRI) and phosrestin II (PRII), are expressed in the photoreceptors of a fruit fly, Drosophila melanogaster. This study presents evidence that the housefly, Musca domestica, also has a protein similar to Drosophila PRI. Our conclusion is based on the following evidence. (1) We identified a Musca photoreceptor protein exhibiting a molecular mass (51 kDa) and an isoelectric point (pI = 8.6) similar to those of Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. (2) Rabbit antibodies raised against Musca PRI, against bovine arrestin, and against a synthetic peptide based on the Drosophila PRI sequence stained the Drosophila and Musca PRIs specifically on 1 and 2-dimensional Western immunoblots. (3) Both Drosophila and Musca PRIs incorporated 32P-radioactivity from gamma-32P-ATP in cell-free homogenates of retinas. Partial peptide digestions of Drosophila and Musca PRIs revealed similarity between these proteins. We observed that Drosophila PRI exists in the random preparation, but it also exists in other subcellular fractions. Immunocytochemistry at the EM level revealed a distribution of both Drosophila and Musca PRI epitopes in membranous vesicular structures in the cytosol as well as in the rhabdomeric microvillar membranes where the visual pigment, rhodopsin, exists. Such distribution of PRI epitopes suggests that PRI and its light-dependent phosphorylation may function in a space remote from the rhabdomere as well as the immediate milieu of photoreception.
Amino Acid Sequence↗
Solubilization and characterization of PGE2 receptor in porcine ciliary epithelium.
PGE2 binding sites or receptors of porcine ciliary nonpigmented epithelial (NPE) and pigmented epithelial (PE) membranes were solubilized with detergents (CHAPS and Triton X100). From the Scatchard plots of PGE2 binding to CHAPS-solubilized proteins, the Kd and Bmax values were calculated to be 35 nM and 470 fmol/mg protein for NPE protein and 65 nM and 430 fmol/mg protein for PE protein, respectively. On the basis of the Kd and Bmax values, the solubilized receptor proteins correspond to PGE2 binding sites of the membranes which have previously been shown to be coupled to adenylate cyclase inhibition. For both NPE and PE proteins, the order of binding potency was PGE2 > PGF2 alpha > PGD2. By gel filtration chromatography of NPE and PE proteins, the molecular mass of the major PGE2 binding peak was estimated to be about 150 KDa when solubilized in CHAPS and 46 KDa for Triton X100 extracts. The Bmax values of membrane-associated binding proteins were increased by GTP, indicating a close association of the PGE2 binding sites with a GTP-binding protein. However, GTP did not affect the Bmax values of detergent-solubilized receptor proteins.
Animals↗
Synthesis of 12(R)- and 12(S)-hydroxyeicosatetraenoic acid by porcine ocular tissues.
Microsomal fractions from porcine ocular tissues synthesized 12(S)-5,8,10,14-hydroxyeicosatetraenoic acid [12(S)-HETE] from arachidonic acid by a membrane-bound lipoxygenase and 12(R)-HETE by the cytochrome P450-dependent monooxygenase system. Both activities were the highest in corneal microsomes. The 12(R)-HETE synthesizing activity of corneal microsomes was dependent on NADPH and inhibited by 0.1 mM SKF-525A, an inhibitor of P450 enzymes. The activity to form 12(R)-enantiomer was significantly enhanced by treatment of corneal epithelium with 3-methylcholanthrene or clofibrate. The induced activity was suppressed by cycloheximide, indicating that the induction of enzyme activities involved a translational process. The effect of these inducers on 12(R)-HETE synthesizing activity appeared to be additive. The activity to form 12(S)-enantiomer was markedly stimulated by 3 mM CaCl2. The 12-lipoxygenase of corneal microsomes was capable of oxygenating linoleic acid in addition to arachidonic acid, a characteristic of 12-lipoxygenases of the leukocyte type. 12(R)-HETE at 10(-6) M inhibited almost completely the Na,K-ATPase of corneal epithelium but had little or no effect on ciliary epithelial enzymic activity. 12(S)-HETE at 10(-6) M also inhibited corneal enzymic activity but to a lesser extent, and had no significant effect on ciliary epithelial Na,K-ATPase activity.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Ultrastructural and immunocytochemical studies of iris vessels in rats with experimental autoimmune uveoretinitis.
In this study, we describe the ultrastructural and immunocytochemical changes that occur in the iris of rats with experimental autoimmune uveoretinitis (EAU). General changes include perivascular infiltration of inflammatory cells, followed by hemorrhage and extensive tissue destruction. Alterations in the iris epithelium were also noted. A breakdown in the blood-iris barrier was demonstrated in some vessels at the peak of inflammation; peroxidase reaction product was seen in the basement membrane and perivascular spaces. We found that, in inflamed iris vessels, endothelial cells and smooth muscle cells become hypertrophic and show increased amounts of synthetic organelles. This finding is similar to our previous observations on endothelial cells and smooth muscle cells in retinal vessels in EAU. In addition, as was reported in the retinal vascular basement membrane in EAU, there is an increase in immunoreactivity of several extracellular matrix molecules in the iris vascular basement membrane; during inflammation, there is a significant increase in immunoreactivity of collagen types I and III, entactin, fibronectin, and laminin. Activated endothelial cells and smooth muscle cells are likely to be involved in the synthesis of certain of these matrix molecules.
Animals↗
Bimodal regulation of adenylate cyclase by prostaglandin E2 receptors in porcine ciliary epithelium.
Prostaglandin E2 (PGE2) binding revealed that porcine ciliary epithelial membranes possess two subclasses of binding sites or receptors. Ciliary nonpigmented epithelial (NPE) membranes have two binding sites (Kd,1 = 35 x 10(-9) M, Bmax,1 = 485 x 10(-12) mol/mg protein; Kd,2 = 0.723 x 10(-6) M, Bmax,2 = 1473 x 10(-12) mol/mg protein), while pigmented epithelial (PE) membranes have one binding site (Kd = 82 x 10(-9) M, Bmax = 377 x 10(-12) mol/mg protein). The three sites of NPE/PE membranes were found to show the highest affinity for PGE2 by competitive binding assay; affinity for PGF2 alpha and PGD2 was several orders of magnitude less. PGE2 binding to the higher affinity site of NPE membranes induced inhibition of adenylate cyclase activity. Activation of the lower affinity site resulted in activation of the cyclase activity. PGE2 binding to PE membranes caused inhibition of adenylate cyclase activity. There is evidence that cyclic adenosine monophosphate (cAMP) affects transport of ions and water in the ciliary epithelium, hence modulates aqueous humor inflow. The present results, therefore suggest that aqueous humor production by the ciliary NPE cells may be influenced by changes in the concentration of PGE2 which binds to the receptor subtypes having opposing effects on adenylate cyclase and regulates intracellular cAMP level.
Adenylyl Cyclases↗
12(R)-hydroxyeicosatetraenoic acid synthesis by 3-methylcholanthrene- and clofibrate-inducible cytochrome P450 in porcine ciliary epithelium.
Porcine ciliary epithelial microsomes synthesized 12[S]-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12[S]-HETE) from arachidonic acid by a membrane-bound lipoxygenase and 12[R]-isomer by the cytochrome P450-dependent monooxygenase system. The activity to form 12(R)-isomer was markedly enhanced by 3-methylcholanthrene and clofibrate. Both basal and induced levels of 12(R)-HETE synthesizing activity were considerably higher in nonpigmented epithelial cells than in pigmented cells of the ciliary processes. The induced activity was suppressed by polyclonal antibodies raised against purified cytochrome P450 IA1 and NADPH-P450 reductase but not by substrates for clofibrate-inducible omega/omega-1 hydroxylases (P450 IVA-mediated). These results suggest that 12(R)-HETE synthesis by porcine ciliary microsomes may be mediated by a cytochrome P450 of the IA family.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Cytochrome P450-mediated prostaglandin omega/omega-1 hydroxylase activities in porcine ciliary body epithelial cells.
The ocular hypotensive effect of topically applied prostaglandins (PGs) is well documented. Although PGs introduced in the posterior chamber accumulate in the anterior tissues (e.g. iris/ciliary complex), little is known about the metabolism of PGs by these tissues. We have recently found that non-pigmented epithelial (NPE) cells and pigmented epithelial (PE) cells are readily separated from porcine ciliary body and cytochrome P450-dependent xenobiotic metabolism is considerably higher in NPE cells than in PE cells. We have therefore investigated in this study the cytochrome P450-mediated PG omega/omega-1 hydroxylase activities of porcine ciliary epithelial cells. The NPE cells show about three times higher activities than do PE cells; the NPE cells, in fact, demonstrate the highest PG omega/omega-1 hydroxylase activities among different ocular tissues. Both omega and omega-1 hydroxylases show broad substrate specificities and hydroxylate PGA1, A2, E1, E2, and lauric acid. The omega/omega-1 hydroxylase activities of NPE and PE, as determined with PGA2 and lauric acid as substrates, are enhanced or induced by treatment of primary cultures of the individual epithelial cells with clofibrate, both activities reaching maximum levels within 48 hr of induction. The induced activities are inhibited almost completely by cycloheximide and actinomycin D. The omega/omega-1 hydroxylase activities of both NPE and PE cells require NADPH and molecular oxygen, are associated with the microsomal fraction, respond to inducers such as clofibrate, and are inhibited by metyrapone and SKF525 A (inhibitors of P450 enzymes). These results support the suggestion that PG omega/omega-1 hydroxylations by NPE and PE are cytochrome P450-mediated reactions.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗