Signal transductions of three isoforms of mouse prostaglandin E receptor EP3 subtype.
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Alternative Splicing↗
Biomedical subjects
T Namba
Publications and source records attributed to T Namba.
Cloning and expression of a cDNA for rat prostacyclin receptor.
A cDNA clone for rat prostacyclin receptor was isolated. The cDNA encodes a protein of 416 amino acid residues (M(r) 44,662) with putative seven transmembrane domains, and belongs to the G protein-coupled receptor superfamily. Specific binding of [3H]iloprost was found in membrane of COS-7 cells transfected with the cDNA (Kd = 1.3 nM) and was displaced with unlabeled prostaglandins in the order of iloprost = cicaprost > PGE1 > STA2 = PGE2 = PGD2 > PGF2 alpha. Northern blot analysis demonstrated that rat prostacyclin receptor mRNA is expressed in the lung, spleen, heart, pancreas, thymus, stomach and aorta.
Amino Acid Sequence↗
Detection of the products of polymerase chain reaction by an ELISA system based on an ion sensitive field effect transistor.
An ELISA system was developed using a pH sensitive ISFET (pH-FET) as a detector, a pipette tip as a solid phase, and urease as a detecting enzyme. Double stranded PCR products with digoxigenin and biotin at both terminals were obtained by using digoxigenin- and biotin-labeled primers. 1 microliters of the PCR solution was directly introduced into the end part of a pipette tip coated with anti-digoxigenin antibody. Biotin-labeled PCR products captured at the solid phase were detected with avidin-urease, of which the activity was measured by a pH-FET in a pH-measuring cell containing urea solution. The assay was used to detect HTLV-I provirus gene integrated in the genome of a human MT-1 cell, and it was found that 100 pg of the genomic DNA of MT-1 cell was specifically detectable after 35 cycles of PCR. Also the detection limit of the present ELISA system itself was determined by using known amounts of purified PCR product labeled with digoxigenin and biotin, and it was found that 10 amol of the labeled DNA in 1 microliter of sample was detectable.
Base Sequence↗
The C-terminus of the prostaglandin-E-receptor EP3 subtype is essential for activation of GTP-binding protein.
Three isoforms of the mouse prostaglandin-E-receptor EP3 subtype (EP3), EP3 alpha, EP3 beta and EP3 gamma, with different C-termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP-binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S. & Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S. & Ichikawa, A. (1993) Eur. J. Biochem. 217, 313-318]. To assess the role of the C-terminus in GTP-binding protein coupling, we truncated the C-terminus of EP3 at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with Gi2, forming an agonist/receptor/Gi2 ternary complex, and to undergo the characteristic conversion of its agonist-binding affinity, mediated by a guanine nucleotide from a low-affinity state to a high-affinity state. However, sulprostone, an EP3 agonist, failed not only to inhibit the forskolin-induced cAMP accumulation in the mutant receptor-expressing cells but also to stimulate the GTPase activity in the mutant receptor-expressing cell membrane. These results indicated that the C-terminus of EP3 is essential for the activation of GTP-binding protein.
Adenylyl Cyclase Inhibitors↗
Molecular cloning and expression of rat prostaglandin E receptor EP2 subtype.
A cDNA clone encoding the rat prostaglandin (PG) E receptor EP2 subtype was cloned from a rat lung cDNA library. It encodes 488 amino acid residues with putative seven-transmembrane domains. Specific binding of [3H]PGE2 was found in COS-7 cells transfected with the cDNA and was displaced with unlabeled prostaglandins in the order of PGE2 = PGE1 >> iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the significant expression being observed in the thymus, lung, spleen, heart stomach, and pancreas.
Amino Acid Sequence↗
Cloning and expression of a cDNA for the human prostacyclin receptor.
A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost >> carbacyclin > prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF 2 alpha did not inhibit it. Iloprost in a concentration-dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.
Amino Acid Sequence↗
cDNA cloning of a mouse prostacyclin receptor. Multiple signaling pathways and expression in thymic medulla.
A functional cDNA for a mouse prostacyclin receptor was isolated from a mouse cDNA library by reverse transcription polymerase chain reaction and hybridization screening. The cDNA encodes a polypeptide of 417 amino acid residues with putative seven transmembrane domains and an calculated molecular weight of 44,722. The amino acid sequence is 30-40% identical in the transmembrane domains to those of the mouse prostaglandin (PG) E receptor subtypes and thromboxane A2 receptor. [3H]Iloprost, a specific prostacyclin receptor radioligand, specifically bound to the membrane of Chinese hamster ovary cells permanently expressing the cDNA with Kd of 4.6 nM. This binding was displaced with unlabeled prostanoids in the order of cicaprost > iloprost, both prostacyclin agonists > PGE1 > carbacyclin >> PGD2 approximately STA2, a thromboxane A2 agonist approximately PGE2 > PGF2 alpha. Iloprost in a concentration-dependent fashion increased cAMP level and generated inositol phosphates in these cells, indicating that the receptor couples to multiple signal transduction pathways. Northern blot analysis revealed that the mRNA is expressed most abundantly in thymus, followed by spleen, heart, and lung. In situ hybridization of thymus showed that it is expressed exclusively in medulla and not in cortex.
Amino Acid Sequence↗
Antiepileptogenic and anticonvulsant effects of NBQX, a selective AMPA receptor antagonist, in the rat kindling model of epilepsy.
To investigate the role of non-NMDA receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline), a potent and selective AMPA receptor antagonist, in the rat kindling model. Systemic administration of 10-40 mg/kg NBQX significantly and dose dependently suppressed previously kindled seizures from the amygdala (AM), assessed in terms of the motor seizure stage and afterdischarge (AD) duration. The maximal effects were observed at 0.5-1 h after drug injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold (GST), the anticonvulsant effects of NBQX on AM-kindled seizures were not reversed, suggesting that the effects were not due to non-specific elevation of the GST. In contrast to AM-kindled seizures, 20-40 mg/kg NBQX significantly suppressed only the motor seizure stage without reducing the AD duration of previously hippocampal-kindled seizures. Daily administration of 15 or 30 mg/kg NBQX prior to each electrical stimulation of the AM markedly and significantly suppressed the development of kindling. During drug sessions, the growth of the AD duration was blocked almost completely, while the waveform of ADs became more complex. These results indicate that NBQX has potent antiepileptogenic and anticonvulsant actions on kindling, at least from the AM and that non-NMDA receptors have an important role in seizure propagation.
Amygdala↗
Cloning and expression of a cDNA for mouse prostaglandin F receptor.
A functional cDNA clone for mouse prostaglandin (PG) F receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of cloned prostanoid receptors, and cross-hybridization screening. The mouse PGF receptor consists of 366 amino acid residues with putative seven transmembrane domains. The sequence revealed the highest homology to the EP1 subtype of PGE receptor and thromboxane (TX) A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGF2 alpha binding. The binding was displaced with unlabeled PGs in the order of PGF2 alpha = 9 alpha, 11 beta PGF2 > PGF 1 alpha > PGD2 > STA2 (a stable TXA2 agonist) > PGE2 > iloprost (a stable PGI2 agonist). PGF2 alpha increased inositol trisphosphate formation in a concentration-dependent manner in COS cells expressing PGF receptor. RNA blot and in situ hybridization analyses demonstrated that the PGF receptor transcripts are abundantly expressed in luteal cells of corpus luteum and in a lesser amount in kidney, heart, stomach, and lung.
Amino Acid Sequence↗
Potassium and caffeine contractures of mouse muscles before and after fatiguing stimulation.
To assess the impairment of muscle membrane excitation, excitation-contraction (E-C) coupling, and contractility during muscle fatigue, we monitored the contracture responses of resting and fatigued muscles on exposure to high potassium and caffeine. On exposure to 140 mmol/L potassium, mouse extensor digitorum longus (EDL) developed a contracture which was 15.7% of tetanic tension before fatigue and 31.7% after fatigue, while soleus developed 59.4% contracture before and 68.8% after fatigue. Potassium causes contractures by depolarizing the muscle fiber membrane. Hence, membrane excitation is reduced in fatigued EDL and soleus. On exposure to 32 mmol/L caffeine, the contracture was 7.1% in resting EDL, 8.5% in fatigued EDL, 50.1% in resting soleus, and 43.7% in fatigued soleus. On exposure to 1 mmol/L caffeine followed by rapid cooling, the contracture was 3.0% in resting EDL, 3.2% in fatigued EDL, 21.5% in resting soleus, and 10.3% in fatigued soleus. Caffeine causes contracture by releasing Ca++ from the sarcoplasmic reticulum. Our results indicate reduced E-C coupling attributable to reduced membrane excitation in fatigued EDL, and reduced contractility in fatigued soleus.
Animals↗
Mapping of the genes encoding mouse thromboxane A2 receptor and prostaglandin E receptor subtypes EP2 and EP3.
Thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) are two of the most representative eicosanoids that are formed from arachidonic acid. They produce a broad spectrum of biological effects mediated through specific cell surface receptors. We have mapped genetic loci for TXA2 receptor, Tbxa2r, and for PGE2 receptor subtypes EP2 and EP3, Ptgerep2 and Ptgerep3, respectively, using restriction fragment length variants in interspecific backcross mice. None of the three loci cosegregated with each other. Tbxa2r mapped to Chr 10, Ptgerep2 mapped to the distal end of Chr 3. Possible human loci for these receptors are predicted based on the homology between mouse and human chromosomes.
Animals↗
Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 in the mouse nervous system.
Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 was investigated by in situ hybridization in the nervous system of the mouse. The hybridization signals for EP3 were widely distributed in the brain and sensory ganglia and specifically localized to neurons. In the dorsal root and trigeminal ganglia, about half of the neurons were labeled intensely. In the brain, intensely labeled neurons were found in Ammon's horn, the preoptic nuclei, lateral hypothalamic area, dorsomedial hypothalamic nucleus, lateral mammillary nucleus, entopeduncular nucleus, substantia nigra pars compacta, locus coeruleus and raphe nuclei. Moderately labeled neurons were seen in the mitral cell layer of the main olfactory bulb, layer V of the entorhinal and parasubicular cortices, layers V and VI of the cerebral neocortex, nuclei of the diagonal band, magnocellular preoptic nucleus, globus pallidus and lateral parabrachial nucleus. In the thalamus, moderately labeled neurons were distributed in the anterior, ventromedial, laterodorsal, paraventricular and central medial nuclei. Based on these distributions, we suggest that EP3 not only mediates prostaglandin E2 signals evoked by blood-borne cytokines in the areas poor in the blood-brain barrier, but also responds to those formed intrinsically within the brain to modulate various neuronal activities. Possible EP3 actions are discussed in relation to the reported neuronal activities of prostaglandin E2 in the brain.
Animals↗
Effects on anti-lipid peroxidation of Cudrania cochinchinensis var. gerontogea.
From the root bark of Cudrania cochinchinensis (Lour.) Kudo et Masamune var. gerontogea (Sieb. et Zucc.) Kudo et Masamune, 3 xanthones, cudraxanthone I, 1,3,7-trihydroxy-2-(3-methylbut-2- enyl)-xanthone, and lancerin, were further isolated and characterized. It was found that pretreatment with 600 mg/kg oral dose of the EtOH root extract of Cudrania cochinchinensis var. gerontogea in mice inhibited the lipid peroxidation stimulated by FeCl2-ascorbic acid-adenosine 5'-diphosphate (ADP) mixture. For searching bioactive constituents, the isolated xanthones from this folk medicine were investigated in anti-lipid peroxidative activities in the rat liver homogenate. The results showed that most of the tested xanthones effectively exhibited anti-lipid peroxidation stimulated by (a) FeCl2-ascorbic acid mixture or (b) CCl4-nicotinamide adenine dinucleotide phosphate (NADPH) mixture. As shown by the result, gerontoxanthone C and I were more active than vitamin E.
Animals↗
Bellidifolin: a potent hypoglycemic agent in streptozotocin (STZ)-induced diabetic rats from Swertia japonica.
Hypoglycemic activity-guided fractionation led to the isolation of five known xanthones and two triterpenoids from the ethyl acetate soluble fraction of Swertia japonica and their identification was based on spectroscopic methods. One of the triterpenes, thysanolactone, was first isolated from this plant. Among the xanthones, bellidifolin showed a potent and dose-dependent hypoglycemic activity in STZ-induced diabetic rats both in i.p. and p.o. administration. A comparative hypoglycemic activity of the other three xanthones together with bellidifolin was also studied.
Animals↗
Energetics of the negative and positive inotropism of pentobarbitone sodium in the canine left ventricle.
OBJECTIVE: Pentobarbitone (sodium) is an anaesthetic widely used in animal experiments. It is known to be a cardiovascular depressant and a coronary dilator, but its effects on myocardial energetics in relation to its negative and positive (due to Gregg's phenomenon) inotropism have not been studied. The aim of this study was therefore to determine whether and how pentobarbitone affects cardiac mechanoenergetics compared with other negative inotropic agents for which data are already available. METHODS: The effects of graded doses of intracoronary pentobarbitone on mechanoenergetics were studied in the excised cross circulated left ventricles of 12 dogs. The framework of the Emax (a contractility index)--VO2 (myocardial oxygen consumption)--PVA (systolic pressure-volume area, a measure of total mechanical energy) relationships was fully utilised. RESULTS: Pentobarbitone increased Emax at low doses in five of the 12 hearts. In two of these five hearts, a marked coronary dilatation was found. Pentobarbitone decreased Emax dose dependently at high doses in all the hearts and lowered the VO2 intercept but not the slope (oxygen cost of PVA) of the VO2-PVA relation. There was no difference in oxygen cost of Emax between pentobarbitone and CaCl2, although they have opposite inotropism. These findings suggest that pentobarbitone depresses myocardial mechanoenergetics via suppression of total calcium handling in the excitation-contraction-relaxation coupling. CONCLUSIONS: Pentobarbitone at low doses partly acts as a positive inotropic agent, but at high doses it acts as a negative inotropic agent like beta blockers and calcium antagonists on cardiac mechanoenergetics in canine blood perfused hearts.
Animals↗
Excitatory changes of rat perirhinal cortex by amygdala-kindling: a study with in vitro slice technique.
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Amygdala↗
Isolation of a human intestinal anaerobe, Bifidobacterium sp. strain SEN, capable of hydrolyzing sennosides to sennidins.
A strictly anaerobic bacterium capable of metabolizing sennosides was isolated from human feces and identified as Bifidobacterium sp., named strain SEN. The bacterium hydrolyzed sennosides A and B to sennidins A and B via sennidin A and B 8-monoglucosides, respectively. Among nine species of Bifidobacterium having beta-glucosidase activity, only Bifidobacterium dentium and B. adolescentis metabolized sennoside B to sennidin B, suggesting that the sennoside-metabolizing bacteria produce a novel type of beta-glucosidase capable of hydrolyzing sennosides to sennidins.
Anthracenes↗
Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney.
Distribution of the mRNAs for three subtypes of prostaglandin E (PGE) receptors in the mouse kidney was investigated by in situ hybridization. The mRNA for EP1 subtype, which is coupled to Ca2+ mobilization, was specifically localized to the collecting ducts from the cortex to the papilla. The mRNA for EP2 subtype, which is linked to stimulation of adenylate cyclase, was localized to the glomeruli. The mRNA for EP3 subtype, which is coupled to inhibition of adenylate cyclase, was located densely in the tubules in the outer medulla and in the distal tubules in the cortex. These results exhibit distinct cellular localization of three subtypes of PGE receptor in the kidney and suggest that PGE2 exerts multiple functions via these subtypes expressed in different segments of the nephron.
Adenylyl Cyclases↗