Toshiko Suzuki-Yamamoto

cDNA cloning, expression and characterization of human prostaglandin F synthase1

A cDNA clone of prostaglandin F synthase (PGFS) was isolated from human lung by using cDNA of bovine lung‐type PGFS as a probe and its protein expressed in Escherichia coli was purified to apparent homogeneity. The human PGFS catalyzed the reduction of prostaglandin (PG) D2, PGH2 and phenanthrenequinone (PQ), and the oxidation of 9α,11β‐PGF2 to PGD2. The k cat/K m values for PGD2 and 9α,11β‐PGF2 were 21 000 and 1800 min−1 mM−1, respectively, indicating that the catalytic efficiency for PGD2 and 9α,11β‐PGF2 was the highest among the various substrates, except for PQ. The PGFS activity in the cytosol of human lung was completely absorbed with anti‐human PGFS antiserum. Moreover, mRNA of PGFS was expressed in peripheral blood lymphocytes and the expression in lymphocytes was markedly suppressed by the T cell mitogen concanavalin A. These results support the notion that human PGFS plays an important role in the pathogenesis of allergic diseases such as asthma.

Localization of 5α‐reductase in the rat main olfactory bulb

The enzyme steroid 5α‐reductase catalyzes the production of dihydroprogesterone and dihydrotestosterone, which were recently recognized as neurosteroids in the brain with variably potential neuroactivity. The present study reports for the first time detailed localization of 5α‐reductase type 1 in the rat main olfactory bulb. The occurrence of 5α‐reductase in the olfactory bulb was detected by reverse transcription‐polymerase chain reaction and Western blotting analyses. In addition, the enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed that 5α‐reductase immunoreactive cells of variable intensity were present in all layers of the olfactory bulb. Multiple immunolabeling revealed that 5α‐reductase was mainly localized in glial cells, namely, in S‐100β‐ and glial fibrillary acidic protein‐immunoreactive astrocytes, 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase)‐immunoreactive oligodendrocytes, and in S‐100β‐ and neuropeptide‐Y‐immunoreactive olfactory ensheathing cells, whereas the bulbar neurons exhibited little immunoreactivity. Quantitative analysis revealed that the number of 5α‐reductase‐immunoreactive cells was greatest in the olfactory nerve layer. The most intense 5α‐reductase‐immunoreactivity was found in the olfactory ensheathing cells, and next in the CNPase‐immunoreactive cells. The 5α‐reductase in the olfactory bulb was expressed constantly throughout different ages and sexes and in neutered and hypophysectomized rats. Thus, 5α‐reductase may contribute via 5α‐reduced metabolites to the formation and maintenance of olfactory inputs and outputs, which were closely associated with the olfactory ensheathing cells and the oligodendrocytes, respectively. J. Comp. Neurol. 493:381–395, 2005.

Identification of prostaglandin F-producing cells in the liver.

Abstract Prostaglandin (PG) F synthase forms PGF2α and 9α, 11β-PGF2 from PGH2 and PGD2, respectively. PGH2 is synthesized from arachidonic acid by cyclooxygenase (COX) and then metabolized to various PGs and thromboxane by specific enzymes. PGD2 is synthesized from PGH2 by PGD synthase. To identify PGF2-producing cells in the rat liver, the occurrence and localization of PGF synthase and COX were studied with immunochemical and immunohistochemical techniques using anti-liver-type PGF synthase and anti-COX antibodies. In Western blot analyses, positive bands of liver-type PGF synthase and constitutive COX-1 were observed at positions approximately 37 kDa and 70–72 kDa, respectively. However, inducible COX-2 was not detected. In the immunohistochemical study, PGF synthase was present in the cytoplasm of the sinusoidal endothelial cells. COX-1 was present on the membranes of the nucleus and endoplasmic reticulum of the endothelial cells and Kupffer cells. Double immunostaining for PGF synthase and COX-1 showed that both enzymes were present in the same endothelial cells. These results suggest that the main site of PGF2 synthesis in the liver is the sinusoidal endothelial cell.

Lack of nociceptin receptor alters body temperature during resting period in mice.

The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL/6J and 129/SV mice, Tcore was continuously recorded under 12:12 h light:dark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity and/or cortisol regulation.

Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord.

Prostaglandin F synthase, producing prostaglandin F(2 alpha) and 9 alpha,11 beta-prostaglandin F(2), has at least two isozymes, lung-type and liver-type ones. The present study including double immunolabelling with microtubule-associated protein 2 indicated that the lung-type isozyme was present in neuronal dendrites and somata of gray matter (relatively intense in lamina I and II in dorsal horn, and IX in ventral horn) and vascular endothelial cells in the rat spinal cord at all segmental levels.

Enhanced hippocampal acetylcholine release in nociceptin-receptor knockout mice

Nociceptin (NOC), an endogenous ligand of the opioid receptor-like 1 receptor, is thought to be involved in learning and memory processes. Since acetylcholine (ACh) is involved in hippocampal function, and the hippocampus plays a critical role on the learning and memory function, hippocampal ACh release in NOC-receptor knockout mice was examined using an in vivo microdialysis method. The release of hippocampal ACh was largely increased in the knockout mice. Furthermore, in the knockout mice, an enhanced hippocampal theta rhythm, which is known to be linked to hippocampal memory function, was also observed. Immunohistochemically, in septum, co-existence of NOC receptor with cholinergic, but not with GABAergic neurons, was verified. The findings demonstrate that the NOC receptor is involved in hippocampal cholinergic function.

Immunocytochemical localization of prostaglandin F synthase II in the rat spinal cord.

Prostaglandin F synthase has at least two isozymes, i.e. prostaglandin F synthase I and II. Recently, we demonstrated immunocytochemically that prostaglandin F synthase I was localized in neuronal dendrites and somata, and in endothelial cells of blood vessels in the whole area of rat spinal cord. In the present study, we immunocytochemically localized prostaglandin F synthase II in ependymal cells and tanycytes surrounding the central canal and in endothelial cells of blood vessels, but not in any neuronal elements at all segmental levels of the rat spinal cord. Immunoelectron microscopy and confocal laser scanning microscopy confirmed these findings and further revealed that strong immunoreactivity was found in the basal processes of the tanycytes. Our present and recent studies using antibodies against the two isozymes of prostaglandin F synthase clearly indicated that they were localized differentially in ependymal (prostaglandin F synthase II) and neuronal elements (prostaglandin F synthase I), but were co-localized in blood vessels in the rat spinal cord. The distinct localization of the two isozymes suggests that prostaglandin F(2) has different transcellular biological actions via different cell groups.

Co-localization of prostaglandin F synthase, cyclooxygenase-1 and prostaglandin F receptor in mouse Leydig cells

In order to promote better understanding of the physiological roles of prostaglandin F2α in the mouse testis, we investigated the protein expression and the cellular localization of the enzymes cyclooxygenase and prostaglandin F synthase that are essential for the production of prostaglandin F2α, and the binding site, which is the prostaglandin F2α receptor (FP). Western blot exhibited the expression of FP protein in wild type mouse testis, and that of prostaglandin F synthase and cyclooxygenase-1 proteins in the both of wild type mouse and FP-deficient mouse testes. The expression of prostaglandin F synthase and cyclooxygenase-1 were detected intensely in Leydig cell-rich fraction, and that of FP was detected equally in Leydig cell-rich fraction and the other fraction. Immunohistochemistry for cyclooxygenase-1 and prostaglandin F synthase demonstrated their co-localization in mouse Leydig cells. Histochemistry for FP demonstrated the localization in Leydig cells and in spermatids of seminiferous tubules. Double histochemical staining confirmed the co-localization of cyclooxygenase-1, prostaglandin F synthase and FP in the Leydig cells. These findings indicate that prostaglandin F2α may have an effect on the functions of Leyding cells in an autocrine fashion. It implies that prostaglandin F synthase and FP are involved in the control of testosterone release from Leydig cells and in spermatogenesis via the local pathway and the hypothalamo-hypophysial-testis pathway, and affect the testicular function.

Cellular localization of 5α-reductase in the rat cerebellum

The enzyme 5α-reductase catalyzes the transformation of progesterone, testosterone, and deoxycorticosterone into 5α-reduced metabolites, which are recognized as neurosteroids in the brain with variable potential neuroactivity. Two isoforms of 5α-reductase were identified in rodents, and, of these, 5α-reductase type 1 (5α-R1) is abundantly expressed in the brain. To understand the multiple influences of neurosteroids in the central nervous system, we need to know their region-specific synthesis. The present study reports the detailed localization of 5α-R1 in the adult rat cerebellum. The occurrence of 5α-R1 was detected by reverse transcription-polymerase chain reaction. The enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed 5α-R1 immunoreactive cells in all cerebellar layers. Multiple immunolabeling revealed that 5α-R1 was mainly localized in glia, such as astrocytes and oligodendrocytes. The most intense immunoreactivity for 5α-R1 was found in Bergmann glia, and the processes of these glia were associated with dendrites of both Purkinje cells and interneurons in the molecular layer. The 5α-R1 in the cerebellum was expressed consistently throughout different ages and sexes, in both gonadectomized and hypophysectomized rats. Thus, 5α-R1 may contribute to the formation and maintenance of the cerebellar neurons through 5α-reduced metabolites, which are synthesized through a complex interaction between neurons and glia.

Roles of Prostaglandin F Synthase

Prostaglandin (PG) F2 is widely distributed in various organs, and exhibits various biological actions such as smooth muscle contraction of uterus [1], bronchus [2], and trachea [3], the initiation of parturition [4], and pain transmission [5]. PGF synthase (PGFS) is a dual function enzyme, which catalyzes the reduction of not only PGD2 but also PGH2 on the same molecule [6]. PGFS exhibits reductase activities toward various carbonyl compounds [6], such as phenanthrenequinone (PQ) in addition to PGD2 and PGH2. PGFS belongs to the aldo-keto reductase (AKR) superfamily based on substrate specificity, molecular weight, and the amino acid sequence [7–10]. Recently, we isolated the clones of four human enzymes [9,10], including PGFS, which belong to the AKR family. Although the amino acid sequences of these enzymes show high homology (83–98%), only PGFS, which was classified to AKR1 C3 [ 10], catalyzed the reduction of PGD2 and PGH2, and the oxidation of 9α, 11β-PGF2 [9, 10]. Moreover, we developed a high-stringency PCR technique to discriminate fine sequence differences using each specific primer, and examined RT-PCR analysis to estimate expression of AKR mRNAs [10]. The tissue distribution of these four enzymes was different, and human PGFS was localized in lung, liver, kidney, muscle, and peripheral blood lymphocyte. The expression in lymphocytes was markedly suppressed by the T cell mitogen concanavalin A. Moreover, PGFS was abundant in the contractile interstitial cells (CIC) in the alveolar septum of bovine lung, and was diffusely present in the cytoplasm of the endothelial cells of bovine liver. These results suggest that PGFS plays important roles in both the physiological and pathophysiological processes.