Mayumi Ujihara

Prostaglandin D2 formation and characterization of its synthetases in various tissues of adult rats.

When the amounts of primary prostaglandins formed from endogenous arachidonic acid were determined in homogenates of various tissues of adult rats, prostaglandin D2 was the major prostaglandin found in most tissues. It was formed actively in the spleen (3100 ng/g tissue/5 min at 25 degrees C), intestine (2600), bone marrow (2400), lung (1100), and stomach (630); moderately in the epididymis, skin, thymus, and brain (140-340); and weakly in other tissues (less than 100). Addition of exogenous arachidonic acid (1 mM) accelerated the formation of prostaglandin D2 in all tissues as follows: spleen (15,000); bone marrow, intestine, thymus, liver, and lung (1600-5200); stomach, adrenal gland, epididymis, brain, salivary gland, skin, spinal cord, and seminal vesicle (380-1000); and other tissues (80-310). The activity of prostaglandin D synthetase (prostaglandin-H2 D-isomerase) was detected in 100,000g supernatants of almost all tissues. As judged by glutathione requirement for the reaction, inhibition of the activity by 1-chloro-2,4-dinitrobenzene, and immunotitration or immunoabsorption analyses with specific antibodies, the enzyme in the epididymis, brain, and spinal cord (1.8-9.2 nmol/min/mg protein) was glutathione-independent prostaglandin D synthetase (Y. Urade, N. Fujimoto, and O. Hayaishi (1985) J. Biol. Chem. 260, 12410-12415). The enzyme in the spleen, thymus, bone marrow, intestine, skin, and stomach (2.0-57.1) was glutathione-requiring prostaglandin D synthetase (Y. Urade, N. Fujimoto, M. Ujihara, and O. Hayaishi (1987) J. Biol. Chem. 262, 3820-3825). The activity in the kidney and testis (3.7-4.5) was catalyzed by glutathione S-transferase. The activity in the liver, lung, adrenal gland, salivary gland, heart, pancreas, and muscle (0.6-5.1) was due to both the glutathione-requiring synthetase and the transferase.

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2α formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).

Tissue manganese levels and liver pyruvate carboxylase activity in magnesium-deficient rats.

To investigate the manganese status in magnesium deficiency, 40 male Wistar rats, 3 wk old, were divided into two groups and fed a magnesium deficient diet or a normal synthetic diet for 2 wk. Dietary magnesium depletion decreased magnesium levels in brain, spinal cord, lung, spleen, kidney, testis, bone, blood, and plasma, while it elevated the magnesium level in liver. In magnesium-depleted rats, calcium concentration was increased in lung, liver, spleen, kidney, and testis, while it was decreased in tibia. In magnesium-depleted rats, manganese concentration was decreased in plasma and all tissues except adrenal glands and blood. Dietary magnesium depletion diminished pyruvate carboxylase (EC 6.4.1.1) activity in the crude mitochondrial fraction of liver. Positive correlation was found between the liver manganese concentration and the pyruvate carboxylase activity. In the magnesium-depleted rats, glucose was decreased while plasma lipids (triglycerides, phospholipids, and total cholesterol) were increased. These results suggest that dietary magnesium deficiency changes manganese metabolism in rats.

Effect of ultraviolet irradiation on the activity of rat skin prostaglandin D synthetase.

The effect of ultraviolet light-B (UVB) irradiation on the activity of prostaglandin (PG) D synthetase was investigated in adult rat skin. Rats were irradiated with 500 mJ/cm2 of UVB, and PGD synthetase activity was determined in 100,000 g supernatant of the homogenate of rat skin in the presence of glutathione (GSH) before and 3, 6, 12, and 24 h after irradiation. The PGD synthetase activity was decreased time dependently, and within 24 h after UVB irradiation it had dropped to 50% of the control level before irradiation. In contrast, the synthesizing activities of PGE2 and PGF2 alpha were unaffected by UVB irradiation. The reduction of PGD synthetase activity after UVB irradiation was much more prominent in the epidermis than in the dermis, which was separated by heat treatment (55 degrees C, 30 sec). Immunohistochemical studies, using anti-(rat spleen PGD synthetase) antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, decreased in the basal layer of the epidermis 24 h after UVB irradiation. These results, together with the reduction of ATPase positive cells in the epidermis after UVB irradiation, suggest that the decrease of PGD synthetase activity in rat skin by UVB irradiation is, at least in part, due to the reduced Langerhans cell population in the basal layer of the epidermis.

ANGIOTENSINOGEN AND KININOGEN: CLONING AND SEQUENCE ANALYSIS OF THE cDNAS

The primary structures of the angiotensinogen precursor and the low molecular weight (LMW) kininogen precursors have been deduced by determining the nucleotide sequences of cloned DNAs complementary to their mRNAs. The angiotensinogen precursor consists of a mature angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites. The LMW kininogen precursors are encoded by two very similar but distinct mRNAs and composed of 436 and 434 amino acid residues. Both kininogens contain two internally homologous sequences in which all amino acid differences between the two kininogens are located. This suggests that these homologous regions may be biologically significant in relation to the existence of two LMW kininogens.

Acantholytic Dyskeratosis on Both Legs

A 33-year-old woman developed pruritic lesions on both legs. There were brownish-red keratotic papules, whose biopsy specimen showed acantholysis and benign dyskeratosis. The duration was approximately 8 years. We considered her as a case of persistent acantholytic dermatosis with atypical distribution.

Changes of the activities of enzymes involved in prostaglandin synthesis in rat skin during development and aging

SummaryThe developmental changes of enzymes involved in prostaglandin (PG) synthesis were investigated in rat skin from birth to 1.5 years old. In all stages of development, the activities of PG-synthesizing enzymes were found in 100,000xg supernatants of homogenates of rat skin, and PGD2 was the major PG among those formed from PGH2 in the presence of 1 mδ glutathione (GSH). The PGD synthetase activity in rat skin at birth was 2.14 nmol/min per mg protein, increasing to the highest level (3.69 nmol/min per mg protein) at 3 weeks after birth and then gradually decreasing up to 1.5 years old. The activities of PGE2 and PGF2α synthetases in rat skin were almost unchanged during development and aging. In contrast, the activity of GSH-S-transferase was at its lowest level at birth and gradually increased, reaching a plateau at 3 weeks after birth and remaining relatively constant during the development. The increase of PGD synthetase activity in 3-week-old rats was mainly due to the increase of specific activity of PGD synthetase in the epidermis, which was separated from the dermis by heat treatment (55° C, 30s). Immunohis-tochemical study, using (rat spleen PGD synthetase)-specific antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, increased in the epidermis in 3-week-old rats. These results suggest that a change of PGD2 synthetase activity during aging of the skin is closely related to the development of ATPase+ Langerhans cells in the epidermis.

Basal levels of prostaglandins in rat skin.

The prostaglandin (PG) represents one of the most important naturally occurring compounds exhibiting substantial biologic activity. Among classic PGs, PGD2 is the major cyclooxygenase product from arachidonic acid, and seems to be an important mediator in acute and chronic inflammation in the skin [4, 10, 12]. In this study, we investigated the basal in situ amounts of PGD2, PGEz, and PGF2~, and the acceleration of their synthesis after incubation with arachidonic acid in rat skin to determine the exact function of PGs in the skin. Male Wistar rats weighing 250-300 g were anesthetized with an intraperitoneal injection of sodium pentobarbital, and their dorsal skin was removed and quickly frozen in liquid nitrogen, as described previously [13]. When the basal levels of prostaglandins were measured by radioimmunoassay, each weighed tissue was directly homogenized in ice-cold ethanol, and when PG levels formed from endogenous arachidonic acid were measured, each weighed frozen tissue was homogenized in 3 vol of 150 mM NaC1 and 10 mM potassium phosphate (pH 7.2), as described previously [13]. The homogenate (1 ml) of whole rat skin in 10 mM phosphate-buffered saline was incubated at 25~ for 5 min, and the incubation was stopped with the addition of 100 lal of 1 N HC1. After addition of [aH]PGD2, PGE2, and PGF2, as a tracer of recovery during the process of extraction, PGs formed in the homogenate were extracted with 10 ml of ice-cold ethanol and purified with a Sep-Pak C18 cartridge [8]. The PG fraction was further submitted to HPLC, as described previously [11, 13]. Finally, the separated

Prostaglandin D Synthetase Is Localized in Antigen Presenting Cells and Mast Cells

Prostaglandin (PG) D2 is a naturally occurring prostanoid that shows various kinds of pharmacological activity, such as inhibition of platelet aggregation, bronchoconstriction, sleep induction, and hypothermia, under various physiological and pathological conditions [1–3]. PGD2 is known to be actively produced by mast cells during IgE stimulation and to modify the anaphylactic process [4].

Prostaglandin D2, a mediator in both immediate and delayed-type immune reactions in the skin.

Kouichi Ikai, MD, Department of Dermatology, Kyoto University, Faculty of Medicine, Yoshida Konoe-machi, Sakyo-ku, Kyoto 606 (Japan) Prostaglandin (PG) D2 is a naturally occurring pros-tanoid that shows various kinds of pharmacological activity such as inhibition of platelet aggregation, bronchocon-striction, sleep induction, and hypothermia under various physiological and pathological conditions [1, 2]. In the skin, however, the in vivo function of PGD2 is not yet fully understood, although PGD2 is the major cyclooxygenase product from arachidonic acid in the epidermis and dermis [2]. In this paper, we report the enzymatic properties and immunohistochemical localization of PGD synthetase in adult rat skin to make clear the biological significance of PGD2 in the skin. Determination of PGD synthetase activity and preparation of anti-rat spleen PGD synthetase antibody were described previously, including the procedure of immuno-histochemistry of PGD synthetase [3, 4]. The activity of PGD synthetase, which isomerizes PGH2 to PGD2, was detected in the 100,000 g supernatant of the homogenate of adult rat skin. The epidermis, which was separated from whole skin by heating (55 °C, 30 s), exhibited about three times higher activity than the dermis. The enzymatic properties of both layers were similar, absolutely glutathione-dependent and completely absorbed by anti-rat spleen PGD synthetase antibody. Immunohistochemical studies using this antibody and the immunoperoxidase method, showed that cytoplasmic distribution of PGD synthetase was prominent in the dendritic cells in adult rat skin. The positively stained dendritic cells were rich in hair follicles. In the dermis, a number of infiltrating cells were immunohistochemically positive for PGD synthetase. By immunoelectron microscopy, positively stained cells in the epidermis showed marked folded nuclei and Birbeck granules with their characteristic rod-shape structure. These cells were considered to be Langerhans’ cells. In the dermis, some of the positive cells showed large nuclei and irregularly projecting narrow cytoplasm with abundant vacuoles and lysosomes. The other positive cells contained oval nuclei and numerous granules in the cytoplasm. The reaction products were also distributed homogeneously in their cytoplasm. The former cells were considered to be macrophages, and the latter, mast cells. These results suggest that PGD2 plays significant roles in immunological functions in the skin, including inflammatory and immediate type immune reactions via mast cells in the dermis, and delayed type immune responses via macrophages in the dermis and Langerhans’ cells in the epidermis.