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Y Urade

Publications and source records attributed to Y Urade.

At least 91 records · Page 5Linked to original sources
PubMed1994

Identification of beta-trace as prostaglandin D synthase.

Human cerebrospinal fluid (CSF) is the richest source of prostaglandin (PG) D synthase, a key enzyme in sleep regulation, having a specific activity of 20-130 nmol/min/mg protein that is almost two orders of magnitude higher than that of crude rat brain homogenate (2-7 nmol/min/mg protein). PGD synthase was purified from human CSF approximately 8-fold to apparent homogeneity in a yield of 6% by ammonium sulfate fractionation followed by column chromatographies on phenyl-Sepharose and DEAE-cellulose. The purified enzyme displayed enzymatic properties almost identical to those of rat brain PGD synthase in terms of Mr, specific activity, optimum pH, Km value, and SH requirement. The purified PGD synthase cross-reacted with an antibody against rat brain PGD synthase and also with that against human beta-trace, a major protein in the CSF. Furthermore, beta-trace cross-reacted with rat brain PGD synthase antibody. The N-terminal sequences of human PGD synthase, beta-trace, and purified PGD synthase from human CSF were essentially identical. These results clearly show that beta-trace is structurally and enzymologically identical to PGD synthase.

Amino Acid Sequence↗
PubMed1994

Two types of endothelin B receptors mediating relaxation in the guinea pig ileum.

In guinea pig ileum, binding assays showed the existence of endothelin (ET) receptors of ETA (isopeptide-selective) and ETB (nonselective) subtypes. ETs induced relaxation followed by contraction. ET-1 induced greater contraction at lower concentrations than ET-3. An ETA antagonist, BQ-123, shifted the concentration-response curves for ETs to the right. An ETB antagonist, IRL 1038, shifted the concentration-response curve for ET-3 to the right and downwards with little effect on the curve for ET-1. In contrast, ET-1 and ET-3 induced relaxation at similar concentrations. The relaxation induced by ETs was composed of an initial transient relaxation followed by sustained relaxation. Only the transient phase was inhibited by IRL 1038 in a concentration-dependent manner. These results suggest that the ET-induced relaxation is mediated by two types of ETB receptor; transient and sustained relaxations are mediated respectively by IRL 1038-sensitive and IRL 1038-insensitive subtypes of ETB receptor. In contrast, the contractile effect seems to be mediated mainly by the ETA receptor and partially by an IRL 1038-sensitive subtype of ETB receptor.

Animals↗
PubMed1994

ETA and ETB receptors on single smooth muscle cells cooperate in mediating guinea pig tracheal contraction.

We investigated the distribution of endothelin A (ETA) and ETB receptors in single smooth muscle cells and their contribution to ET-induced contractions of guinea pig trachea. ETA and ETB receptors were detected in smooth muscle membranes (maximum binding capacities of 810 and 360 fmol/mg protein and dissociation constants of 38 and 5.1 pM for 125I-labeled ET-1 and 125I-ET-3, respectively) and visualized autoradiographically in primary cultured cells. ET-1 and ET-3 evoked concentration-dependent increases in intracellular Ca2+ concentration and smooth muscle tension. The half-maximally effective concentrations of ET-1 and ET-3 at inducing contractions were 1.9 and 2.7 nM, respectively. The Ca2+ responses showed tachyphylaxis to both ETs after stimulation with ET-1, but only to ET-3 after stimulation with ET-3. Consecutive applications of ET-3 and ET-1 (10 nM each) classified the cells into ETA dominant (approximately 30%) responding to only ET-1, ETB dominant (approximately 20%) responding to only ET-3, and ETA- and ETB-possessing (approximately 50%) cells responding to both. The ETA antagonist, 10 microM BQ-123, attenuated ET-1-induced contractions but did not affect the ET-3-induced contractions. The results indicate that both receptors coexist in a major population of smooth muscle cells and cooperate in mediating ET-1-induced contractions.

Animals↗
PubMed1993

Dominant expression of mRNA for prostaglandin D synthase in leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain.

Glutathione-independent prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z,13E)-(15S)-9 alpha,11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase, EC 5.3.99.2] is an enzyme responsible for biosynthesis of prostaglandin D2 in the central nervous system. In situ hybridization with antisense RNA for the enzyme indicated that mRNA for the enzyme was predominantly expressed in the leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain. The findings agree with those obtained by immunohistochemical staining with antibodies against the enzyme. It was further revealed that prostaglandin D synthase activity was considerably greater in the isolated leptomeninges (14.2 nmol per min per mg of protein) and choroid plexus (7.0 nmol per min per mg of protein) than the activity in the whole brain (2.0 nmol per min per mg of protein). These results, taken together, indicate that the enzyme is mainly synthesized and located in the leptomeninges, choroid plexus, and oligodendrocytes in the brain.

Animals↗
PubMed1993

Contraction of smooth muscle by activation of endothelin receptors on autonomic neurons.

Endothelin receptors, predominantly of the ETB type, were localized to cell bodies, processes, and varicosities of cholinergic and adrenergic intramural autonomic neurons that were present in primary cultures of guinea pig tracheal smooth muscle. Stimulation of the neuronal ETB receptor produced a tetrodotoxin-sensitive increase in the intracellular calcium concentration in neurons which was followed by contraction of the neighboring smooth muscle cells. These observations suggest that endothelins can induce smooth muscle contraction by means of a neuronally mediated mechanism, in addition to their direct actions on the smooth muscle.

Animals↗
PubMed1993

ETB receptor antagonist, IRL 1038, selectively inhibits the endothelin-induced endothelium-dependent vascular relaxation.

In isolated rat aorta, endothelin-1 induced contractions at lower concentrations than endothelin-3. The contractile effects were augmented by removing the endothelium. In contrast, endothelium-1 and endothelin-3 at similar concentrations induced endothelium-dependent relaxation in norepinephrine-stimulated aorta. IRL 1038 ([Cys11,Cys15]endothelin-1(11-21); 3 microM) augmented the contractile effects of endothelins only in the presence of the endothelium. IRL 1038 (0.3-3 microM) inhibited the endothelium-dependent relaxation induced by endothelins but not by carbachol. IRL 1038 itself did not change muscle tension. These results suggest that IRL 1038 is a novel antagonist of the ETB receptor responsible for the release of relaxing factor from the vascular endothelium.

Animals↗
PubMed1993

A novel subtype of endothelin B receptor mediating contraction in swine pulmonary vein.

Effects of agonists and antagonists of endothelin (ET) receptors were examined in swine pulmonary artery and vein and in rabbit saphenous vein. ET-1, but not ETB receptor agonists, sarafotoxin S6c (STXc) and IRL 1620, induced contraction in pulmonary artery. This effect was inhibited by the ETA receptor antagonists, BQ-123 and FR139317, but not by the ETB receptor antagonist, IRL 1038. Pulmonary artery precontracted by norepinephrine was relaxed by ET-3 in an endothelium-dependent manner. This relaxation was inhibited by IRL 1038 but not by BQ-123. In pulmonary vein, ET-1, ET-3, STXc and IRL 1620 induced contractions at a similar concentration range. ET-1 induced contraction also in saphenous vein. These contractions were not inhibited by BQ-123, FR139317 or IRL 1038. These results suggest that the isopeptide-selective ETA receptor mediates contraction in swine pulmonary artery whereas the isopeptide-nonselective ETB receptor mediates release of endothelium-derived relaxing factor. In contrast, contractions in the veins may be mediated by a novel subtype of isopeptide-nonselective ETB receptor which is not inhibited by IRL 1038.

Animals↗
PubMed1993

Induction of endothelium-dependent relaxation in the rat aorta by IRL 1620, a novel and selective agonist at the endothelin ETB receptor.

1. The effects of a novel and selective agonist at the endothelin ETB receptor, IRL 1620 (Suc-[Glu9, Ala11,15] endothelin-1 (8-21)), were examined in the isolated aorta of the rat. 2. IRL 1620 (1-300 nM) changed neither the resting tone nor the cytosolic Ca2+ level ([Ca2+]i) of the aorta without endothelium. In the presence of endothelium, however, IRL 1620 increased endothelial [Ca2+]i with little effect on the muscle tone. In the absence of external Ca2+, IRL 1620 still induced a transient increase in endothelial [Ca2+]i. 3. Noradrenaline (100 nM) increased both muscle [Ca2+]i and tension. IRL 1620 (1-300 nM) relaxed the muscle with an increase in endothelial [Ca2+]i only in the presence of endothelium. An inhibitor of nitric oxide synthase, 100 microM NG-monomethyl-L-arginine, inhibited the relaxant effect of IRL 1620 but not the increase in endothelial [Ca2+]i. 4. In resting and noradrenaline-stimulated aorta, the effects of IRL 1620 were inhibited by a selective antagonist of the ETB receptor, IRL 1038 (0.3-3 microM), although a selective antagonist of the ETA receptor, BQ-123 (3 microM), was ineffective. Verapamil (10 microM) did not alter the effects of IRL 1620. 5. A muscarinic receptor agonist, carbachol (1 microM), also induced endothelium-dependent relaxation with an increase in endothelial [Ca2+]i. However, the effects of carbachol were not inhibited by the ETB antagonist, IRL 1038 (3 microM). 6. These results suggest that IRL 1620 is a selective agonist at the ETB receptor which increases endothelial [Ca2+]i by releasing Ca2+ from storage sites and by opening non-L type Ca2+ channels,activates nitric oxide synthase, releases nitric oxide, and relaxes vascular smooth muscle.

Animals↗
PubMed1993

A selective agonist of endothelin type B receptor, IRL 1620, stimulates cyclic GMP increase via nitric oxide formation in rat aorta.

The signal transduction pathways of endothelin (ET)-induced vasorelaxation in rat aorta were investigated. An agonist for ETB receptors, IRL 1620, induced transient increases in cytosolic Ca++ (peak at about 10 sec) and cyclic GMP (peak at about 20 sec) accompanied by transient vasorelaxation (peak at about 60 sec) in aortic strips precontracted with 100 nM norepinephrine. The cyclic GMP content was increased 3- to 6-fold from the basal level (1.6 +/- 0.2 fmol/micrograms of protein) with 1 nM to 1 microM IRL 1620. The cyclic GMP elevation was endothelium-dependent, abolished in the presence of 100 microM NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, and recovered after the further addition of 1 mM L-arginine. An ETB receptor antagonist, IRL 1038 (3 microM), inhibited completely the cyclic GMP increase induced by 100 nM IRL 1620 (8.1 +/- 0.6 fmol/micrograms of protein) without affecting the basal level. On the other hand, an ETA receptor antagonist, 3 microM BQ-123, enhanced significantly both the basal level (3.7 +/- 0.6 fmol/micrograms of protein) and the IRL IRL 1620-induced production (12.2 +/- 0.8 fmol/micrograms of protein) of cyclic GMP. Specific binding sites for [125I]IRL 1620 were detected in rat aortic membranes with a dissociation constant of 37.0 pM and maximal binding capacity of 36.6 fmol/mg of protein, which disappeared after removing the endothelium. Unlabeled ET-1, ET-3, IRL 1620 and IRL 1038, but not BQ-123, displaced the binding of [125I]IRL 1620 with inhibitory constants of 38.5 pM, 36.6 pM, 97.8 pM and 8.9 nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1992

An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11-21)

In the inhibition of specific binding of [125I]endothelins (ETs) to membrane from various tissues of rats, guinea pigs, pigs and humans, [Cys11-Cys15]-ET-1(11-21), IRL 1038, has a much higher affinity for ETB receptors (Ki = 6-11 nM) than for ETA receptors (Ki = 0.4-0.7 microM). In contraction assays, with ET-3 as a stimulant, 3 microM IRL 1038 antagonized the ETB receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is therefore, considered to be the first antagonist selective to the ETB receptor.

Amino Acid Sequence↗
PubMed1992

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.

Animals↗
PubMed1992

Structural organization of the gene for prostaglandin D synthase in the rat brain.

A 3-kilobase-pair gene for rat brain prostaglandin D synthase [(5Z,13E)-(15S)-9 alpha,11 alpha-epidoxy-15-hydroxyprosta-5,13- dienoate D-isomerase, EC 5.3.99.2], which belongs to the lipocalin family, was isolated from a rat genomic DNA library by plaque hybridization with the cDNA for the enzyme. The gene contains seven exons, and all the splice donor and acceptor sites conform to the GT/AG rule. Transcription initiates at a guanine residue 39 base pairs upstream of the translation initiation codon, as determined by primer-extension analysis of rat brain mRNA. The 5'-flanking region of the gene lacks typical transcriptional regulatory sequences, such as TATA and CAAT boxes, but contains several sets of inverted repeats, direct repeats, and sequences resembling the transcriptional factor Sp1-binding site. The gene structure of prostaglandin D synthase is remarkably analogous to those of other lipocalins, such as beta-lactoglobulin, alpha 2-urinary globulin, placental protein 14, and alpha 1-microglobulin, in terms of number and sizes of exons and phase of splicing of introns. Furthermore, in a multiple alignment of the deduced amino acid sequences, positions of exon/intron junction of the prostaglandin D synthase gene are highly conserved and located around the positions of those of the genes for other lipocalins despite a weak homology.

Amino Acid Sequence↗
PubMed1992

A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor.

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.

Amino Acid Sequence↗
PubMed1992

Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells.

Embryonic bovine tracheal (EBTr) cells were found to possess receptors for endothelin (ET) of ET-1-selective (ETA) subtype with a Kd for ET-1 of 114 pM and a Bmax of 12.9 fmol/10(5) cells. Stimulation of EBTr cells with 100 pM to 100 nM ET-1 increased the contents of both inositol phosphates and cAMP in a concentration-dependent manner, indicating that the receptors are coupled to both phosphatidylinositol hydrolysis and cAMP formation in EBTr cells.

Animals↗
PubMed1992

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein.

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin 1 (ET-1) actively. Specific binding of [125I]ET-1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET-1. However, addition of phosphoramidon, an inhibitor of ET-converting enzyme, to the medium reduced the production of ET-1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET-1. Binding studies using phosphoramidon-treated HUVECs indicated the existence of a non-isopeptide-selective type (ETB) of ET receptor with a Kd of 17 pM. This receptor is thought to be involved in ET-induced vasodilation in an autocrine manner in vivo.

Binding Sites↗
PubMed1992

Biochemical and immunohistochemical demonstration of a tightly bound form of prostaglandin E2 in the rat brain.

Basal levels of prostaglandin E2 in the rat brain were determined by radioimmunoassay to be 0.68-0.79 pmol/g brain. About one-third of the prostaglandin E2 (0.23-0.28 pmol/g) was resistant to extraction with ethanol, but could be recovered with a mixture of ethanol and 1 N HCl (9:1, v/v), indicating that a tightly bound form of prostaglandin E2 exists in the brain. The amount of the bound form of prostaglandin E2 was almost unchanged by pentylenetetrazole-induced convulsion or by transcardial perfusion with a formaldehyde solution, although these treatments resulted in 40- to 80-fold increases in prostaglandin E2 content extracted with ethanol at neutral pH. A polyclonal antibody against prostaglandin E2-albumin conjugates recognized the bound form of prostaglandin E2, giving a punctate appearance in many neuronal cell bodies in the brain. Although almost all of the neuronal perikarya were immunoreactive for prostaglandin E2, intense immunoreactivity was observed in the mitral cell layer of the olfactory bulb, layer V of the cerebral neocortex, anterodorsal and reticular nuclei of the thalamus, supraoptic, paraventricular, accessory neurosecretory and lateral mammaillary nuclei of the hypothalamus, mesencephalic trigeminal nucleus, nucleus of the trapezoid body and deep cerebellar nuclei. When the cerebral neocortical regions were observed electron microscopically, immunoreaction products were seen as fine granules which were clustered into small patches in the cytoplasm of neuronal cell bodies and proximal dendrites. No immunoreaction products were seen in glial cells or endothelial cells. These results suggest that prostaglandin E2 is involved in fundamental processes of neurons.

Animals↗