Biomedical subjects

T Namba

Publications and source records attributed to T Namba.

At least 145 records · Page 8Linked to original sources

PubMed1994

Molecular cloning of human prostacyclin receptor cDNA and its gene expression in the cardiovascular system.

BACKGROUND: Prostacyclin elicits a potent vasodilation and inhibition of platelet aggregation through binding to its membrane receptor. The impairment of prostacyclin receptor activity is implicated in various human cardiovascular diseases. In the present study, we succeeded in the isolation and characterization of human prostacyclin receptor cDNA and elucidated its gene expression in human tissues. METHODS AND RESULTS: We isolated a cDNA clone encoding the human prostacyclin receptor from a human lung cDNA library. The isolated cDNA clone encodes a 386-amino acid protein with seven putative transmembrane domains, which belongs to the G protein-coupled receptor superfamily. [3H]iloprost, a prostacyclin receptor agonist, specifically bound to the receptor transiently expressed in COS-7 cells. The binding was inhibited in the rank order of iloprost = cicaprost, another prostacyclin receptor agonist, > prostaglandin E1 (PGE1) >> PGE2, PGF2 alpha, PGD2, STA2. In addition, iloprost dose-dependently stimulated cAMP generation in these COS-7 cells. These results are consistent with the characteristics of the human prostacyclin receptor. Northern blotting analysis on human tissues revealed that prostacyclin receptor mRNA is abundantly expressed in the aorta, lung, atrium, ventricle, and kidney. CONCLUSIONS: We cloned human prostacyclin receptor cDNA and elucidated its abundant gene expression in the human cardiovascular system. The present study will lead to better understanding of the significance of prostacyclin in humans and further facilitate the clinical application of prostacyclin.

Amino Acid Sequence↗

PubMed1994

Enzymatic sulfation of glycosides and their corresponding aglycones by arylsulfate sulfotransferase from a human intestinal bacterium.

A novel type of arylsulfate sulfotransferase (ASST) from a predominant human intestinal bacterium catalyzes the stoichiometric transfer of a sulfate group from phenolic sulfate esters to phenols. We clarified that polyphenols were better substrates of this enzyme than the corresponding glycosides. Additionally, a coumarin derivative, esculetin, was sulfated by ASST at the 6-position to give 6-monosulfate. Therefore, ASST is more useful for the preparation of sulfated polyphenols at their specific hydroxyl groups and would play an important role in the metabolism of phenolic compounds in vegetable food and traditional medicines.

Arylsulfotransferase↗

PubMed1994

Matteuorienate A and B, two new and potent aldose reductase inhibitors from Matteuccia orientalis (Hook.) Trev.

Matteuorienate A and B, two new C-methyl flavanone derivatives, were isolated from the Matteuccia orientalis (Hook.) Trev. (Aspidiaceae), and their structures were determined by the use of spectroscopic methods including 2D-NMR (1H-1H COSY, 1H-13C COSY and 1H-13C long-range COSY) experiments and chemical methods. Both the compounds were found to be very strong inhibitors of aldose reductase among the natural products.

Aldehyde Reductase↗

PubMed1994

Estimation of total carbon dioxide contents in canine coronary arterial and venous whole blood samples.

We examined whether total carbon dioxide content (CCO2) estimation equations for human whole blood in the literature are applicable to canine coronary arterial and venous blood samples. PCO2 of the tested blood samples covered 19-52 mmHg; PO2 19-398 mmHg. Three CCO2 estimation equations developed by Kelman, Godfrey, and Douglas et al yielded CCO2 values over a range of 26.7-54.9 vol%, where plasma CCO2 values calculated by the Henderson-Hasselbalch equation ranged over 27.7-62.2 vol%. The estimated blood CCO2 values were almost identical among the three equations. They also closely correlated with plasma CCO2 values measured after hemolysis with saponin. We conclude that the human whole blood CCO2 estimation equations are applicable to canine coronary arterial and venous blood samples. These CCO2 equations may be used for canine cardiac metabolic studies.

Animals↗

PubMed1993

Opposite coupling of prostaglandin E receptor EP3C with Gs and G(o). Stimulation of Gs and inhibition of G(o).

We recently identified four isoforms of bovine prostaglandin E receptor EP3 subtype, which are coupled to different signaling pathways; EP3A is coupled to inhibition of adenylate cyclase, while EP3B and EP3C are coupled to its stimulation and EP3D is coupled to phosphatidylinositol turnover, in addition to the adenylate cyclase system (Namba, T., Sugimoto, Y., Negishi, M., Irie, A., Ushikubi, F., Kakizuka, Ito, S., A., Ichikawa, A., and Narumiya, S. (1993) Nature 365, 166-170). We examined here the identity of coupled G proteins and their regulation by one of the isoforms, EP3C, in the membranes of EP3C cDNA-transfected Chinese hamster ovary cells. M&B 28767, an EP3 agonist, stimulated the GTPase activity in the pertussis toxin (PT)-treated cell membrane, but inhibited it in the cholera toxin (CT)-treated cell membrane, while the agonist neither stimulated nor inhibited it in the both PT- and CT-treated cell membrane. In the PT- and CT-treated cell membrane reconstituted with various G proteins, M&B 28767 inhibited the GTPase activity of G(o), but stimulated that of Gs. On the other hand, M&B 28767 did not affect the GTPase activity of Gi1, Gi2, or Gi3. M&B 28767 increased the apparent affinity of G(o) for GDP without any change in that for GTP, as assessed by displacement of [35S]GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to G(o). In contrast, M&B 28767 increased the apparent affinity of Gs for GTP but decreased that for GDP. These results demonstrated that the EP3 receptor isoform is coupled to two different G proteins, and oppositely regulates their activities, inhibition of G(o), and stimulation of Gs.

Alprostadil↗

PubMed1993

Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis.

We have recently revealed that the thymus is the organ showing the highest expression of thromboxane (TX) A2 receptor in mice. In this study, thymic cell populations expressing the receptor were identified, and the effects of a TXA2 agonist on these cells were examined. Radioligand binding using a TXA2 receptor-specific radioligand revealed a single class of binding sites in the thymocytes with an affinity and specificity identical to those reported for the TXA2 receptor. The receptor density in these cells was comparable to that seen in blood platelets. This receptor is most highly expressed in CD4-8- and CD4+8+ immature thymocytes, followed by CD4+8- and CD4-8+ cells. The receptor density in splenic T cells was less than one fifth of that in CD4+8+ cells and no binding activity was detectable in splenic B cells. The addition of a TXA2 agonist, STA2, to thymocytes induced the disappearance of the CD4+8+ cells in a time- and concentration-dependent manner and caused DNA fragmentation. These changes were blocked by a specific TXA2 antagonist, S-145. These results demonstrate that TXA2 induces apoptotic cell death in immature thymocytes by acting on the TXA2 receptor on their cell surface and suggest a role for the TXA2/TXA2 receptor system in the thymic micro-environment.

Animals↗

PubMed1993

Third isoform of the prostaglandin-E-receptor EP3 subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase.

A functional cDNA clone for a third isoform of the mouse prostaglandin-E-receptor EP3 subtype, derived by alternative RNA splicing, named the EP3 gamma receptor, was obtained in addition to those for the two other isoforms, EP3 alpha and EP3 beta. The three isoforms are only different in the amino acid sequence of the putative cytoplasmic carboxy-terminal tail. When expressed, EP3 gamma shows identical ligand-binding properties to these of the other isoforms. The EP3-selective agonist, M&B 28767, increased the basal cAMP level and inhibited the forskolin-induced increase in the cAMP level in EP3 gamma, while it decreased both the basal and forskolin-elevated cAMP levels in EP3 alpha and EP3 beta. The M&B 28767-stimulated GTPase activity consisted of pertussis-toxin-sensitive and cholera-toxin-sensitive portions in the EP3 gamma-expressing cell membrane, suggested that EP3 gamma is coupled to both guanine nucleotide-binding inhibitory and stimulatory proteins. These results indicate that EP3 gamma is coupled to both stimulation and inhibition of adenylate cyclase, but that EP3 alpha and EP3 beta are exclusively coupled to inhibition of adenylate cyclase. Thus, alternative splicing produces a third isoform with a different carboxy-terminal tail, which differs from the other two isoforms in the specificity of coupling to a signal-transduction pathway.

Adenylyl Cyclase Inhibitors↗

PubMed1993

Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

Amino Acid Sequence↗

PubMed1993

Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity.

Peptide hormones, neurotransmitters, and autacoids activate a family of seven-transmembrane-domain receptors. Each of these receptors specifically couples to one of several G proteins, Gs, Gi, G(o) and Gp, to activate a specific second messenger system. Cell surface receptors for prostanoids have been characterized pharmacologically and the complementary DNAs for thromboxane A2 receptor and the EP3 subtype of the prostaglandin (PG)E receptor reveal that they belong to the seven-transmembrane-domain receptor family. The EP3 receptor mediates the diverse physiological actions of PGE2 (ref. 3). Although most of them occur through coupling of the EP3 receptor to Gi and inhibition of adenylyl cyclase, the EP3-mediated contraction of uterine muscle can only occur by activation of another second messenger pathway. In chromaffin cells, two different second messenger pathways are activated by PGE2 binding to an apparently single EP3 receptor class. Here we show that at least four isoforms of the EP3 receptor, which differ only at their C-terminal tails and are produced by alternative splicing, couple to different G proteins to activate different second messenger systems.

Adrenal Medulla↗

PubMed1993

Characterization of the prostaglandin E receptor expressed on a cultured mast cell line, BNu-2cl3.

Interleukin 3-dependent BNu-2cl3 mast cells, mucosal type-like mast cells, exhibited a specific high-affinity binding site for [3H]prostaglandin (PG) E2. The binding was completely displaced by M&B 28767, an EP3-selective agonist, but not by EP1- or EP2-selective ligands, indicating that the PGE2 binding site is of the EP3 subtype PGE receptor. Whereas the EP3 subtype is presumed to be coupled to inhibition of adenylate cyclase in various tissues and cells, in BNu-2cl3 cells PGE2 had no ability to inhibit adenylate cyclase activity, while it induced concentration-dependent stimulation of phosphoinositide metabolism and caused an increase in the intracellular free Ca2+ concentration in a pertussis toxin-sensitive manner. PGE2 by itself did not evoke histamine release from the cells, but it markedly stimulated histamine release in concert with ionomycin, a Ca2+ ionophore. The PGE2-stimulated release was also completely blocked by pertussis toxin. Thus, the PGE receptor expressed on BNu-2cl3 mast cells is of the EP3 subtype and is linked to phosphoinositide metabolism via a pertussis toxin-sensitive G protein, and this activation leads to histamine release.

Adenylate Cyclase Toxin↗

PubMed1993

Adenosine inhibits efferent function of extrinsic capsaicin-sensitive sensory nerves in the enteric nervous system.

Capsaicin (1-3 microM) and electrical stimulation of mesenteric nerves in the presence of hexamethonium and guanethidine antidromically stimulate extrinsic sensory nerve fibers to produce a specific slow depolarizing response of myenteric neurons and a contractile response of muscles in the isolated guinea-pig ileum, mediated by release of substance P and acetylcholine. Adenosine (1-100 microM) inhibited the response to mesenteric nerve stimulation. Adenosine (10-100 microM) suppressed the contractile response to a threshold concentration of capsaicin (1 microM) while leaving the contractile response to a submaximal concentration of substance P (1 nM) and acetylcholine (0.1 microM) intact. Adenosine (1-10 microM) inhibited dose dependently the capsaicin 10 microM)-induced depolarization of myenteric neurons, but did not inhibit the depolarizing response to exogenous substance P. The adenosine P1 receptor antagonist, 8-phenyltheophylline (1-10 microM), antagonized the inhibitory effect of adenosine (1-10 microM) on the mechanical responses. We conclude that adenosine-induced prejunctional inhibition of the mechanical responses is mediated by adenosine P1 receptors.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG 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, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

Amino Acid Sequence↗

PubMed1993

Functional interaction of prostaglandin E receptor EP3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding.

The functional interaction of prostaglandin E (PGE) receptor EP3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP3 receptor cDNA-transfected Chinese hamster ovary cells. PGE2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE2 binding to the membranes was increased by GTP gamma S, and PT treatment also increased the binding activity to the same level as that increased by GTP gamma S, but the sensitivity of GTP gamma S was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > GO, but GTP gamma S completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in Kd without the change of Bmax, and due to suppression of association rate. [3H]PGE2-bound EP3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTP gamma S was eluted at the position of M(r) = approx. 60 kDa, similar to the relative molecular mass of EP3 receptor deduced from its amino acid sequence. In contrast, [3H]PGE2-bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M(r) = approx. 130 kDa, corresponding to the M(r) of the complex of EP3 receptor and Gi2, but GTP gamma S shifted the position of its elution from M(r) = 130 to 60 kDa. Furthermore, addition of PGE2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > GO. These results indicate that EP3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

Adenosine Diphosphate↗

PubMed1993

Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins.

Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 subtype derived from alternative RNA splicing were obtained. The two isoforms are only different in the sequence of the putative cytoplasmic carboxyl-terminal tail and their hydrophobicity; one isoform, named EP3 alpha, has a hydrophilic tail, and the other, named EP3 beta, has a hydrophobic tail. When expressed, the two receptors displayed identical ligand binding properties but different responses to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Without a change in the Bmax value, GTP gamma S increased Kd for prostaglandin E2 of EP3 beta and decreased that of EP3 alpha. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of Gi2. Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3 alpha than EP3 beta for 50% inhibition of cAMP formation. A similar difference in agonist potency was observed also for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails show different coupling to the Gi protein, leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins.

Adenylyl Cyclases↗

PubMed1993

Mechanisms of fatigue in normal intercostal muscle and muscle from patients with myasthenia gravis.

Fatigue mechanisms in normal intercostal muscle and muscle from patients with myasthenia gravis (MG) were evaluated by monitoring the compound muscle action potential (CMAP) and tetanic tension responses to repetitive nerve or muscle stimulation in vitro. When fatigue was induced by nerve stimulation at 30 Hz for 0.5 s every 2.5 s, about half of the original tension decreased after 30 min in normal muscle and 5 min in MG muscle. Analysis of the changes in area of CMAPs and tension indicated that impairment of neuromuscular transmission, muscle membrane excitation, and excitation-contraction (E-C) coupling and contractility accounted for 40%, 29%, and 31% of fatigue in normal muscle, and 83%, 0%, and 17% of fatigue in MG muscle. When fatigue was induced by muscle stimulation at 30 Hz, tension declined by a quarter after 30 min in normal muscle, but by a half after 17 min in MG muscle. Impairment of muscle membrane excitation and E-C coupling and contractility accounted for 58% and 42% of fatigue in normal muscle, and 22% and 78% of fatigue in MG muscle. Thus, fatigue of normal muscle is caused by impairment of at least four processes, and enhanced fatigue of MG muscle is caused by greater impairment of neuromuscular transmission, E-C coupling, and contractility.

Action Potentials↗

PubMed1993

Effect of cocaine on leakage of creatine kinase from isolated fast and slow muscles of rat.

Since patients with cocaine overdose were reported to develop rhabdomyolysis involving skeletal muscle damage leading to elevated levels of serum creatine kinase (CK), we determined whether cocaine can directly act on isolated rat skeletal muscles and increase the leakage of CK. In the fast-twitch muscle such as the extensor digitorum longus (EDL), following exposure to normal physiological solution for 1, 2, 3, and 4 hr, the mean leakage of CK was 0.6, 0.7, 0.9, and 1.2 units/mg of muscle respectively. On exposure of EDL to 0.1, 0.5, and 1.0 mM cocaine, there was no significant change in CK leakage. In the slow-twitch muscle such as the soleus, following exposure to normal physiological solution for 1, 2, 3, and 4 hr, the mean leakage of CK was 1.5, 2.2, 2.7, and 3.1 units/mg, which was significantly greater (P < 0.001) than in EDL at each time interval. On exposure of soleus to 0.1 mM cocaine, the CK leakage did not increase significantly, but on exposure to 0.5 mM cocaine, it significantly increased to 2.4, 3.4, 4.4, and 5.7 units/mg, and on exposure to 1.0 mM cocaine, it further increased to 2.7, 4.9, 6.5, and 7.6 units/mg. The CK activity of fresh muscle homogenate was 115.5 units/mg in EDL and 51.9 units/mg in soleus. These results indicate that cocaine can directly act on skeletal muscle and increase the leakage of CK especially from slow-twitch muscle like soleus, but not from fast-twitch muscle like EDL.

Animals↗

PubMed1993

Antiepileptogenic action of 7-chlorokynurenic acid on amygdala kindling of rats.

To investigate the role of strychnine-insensitive glycine receptors in epilepsy, we studied the effects of 7-chlorokynurenic acid (7-CK), a selective strychnine-insensitive glycine receptor antagonist, on amygdala kindling development and previously amygdala-kindled seizures in rats. ICV administration of 7-CK (10 or 20 micrograms) suppressed amygdala kindling development, according to the motor seizure stage and afterdischarge development, in a dose-dependent manner. However, 7-CK had no significant effect on previously kindled seizures at either of these doses nor did 20 micrograms at any time (15 min, 30 min, 2 h, and 24 h) after injection studied. These results demonstrate that this selective strychnine-insensitive glycine receptor antagonist has antiepileptogenic activity and suggest a role for the glycine receptors in the contribution of the NMDA receptor complex to epileptogenic events.

Amygdala↗

PubMed1993

Antiviral traditional medicines against herpes simplex virus (HSV-1), poliovirus, and measles virus in vitro and their therapeutic efficacies for HSV-1 infection in mice.

One hundred forty-two kinds of traditional medicines, which have been historically used in China, Indonesia, and Japan, were examined for the antiviral activity of their hot water (HW) extracts against herpes simplex virus type 1 (HSV-1), poliovirus type 1, and measles virus by plaque reduction assay. Thirty-two, 55, and 30 HW-extracts of them showed anti-HSV-1, antipoliovirus, and anti-measles virus activities, respectively. Among the 32 HW-extracts with anti-HSV-1 activity, 3 HW-extracts had anti-HSV-1 activity alone and the others showed anti-HSV-1 activity with anti-poliovirus and/or anti-measles virus activities. The 32 HW-extracts were further examined for their therapeutic efficacies of HSV-1 infection in mice. The mice were infected cutaneously with HSV-1 and HW-extracts were orally administered three times daily. Twelve HW-extracts, currently used for the treatment of various diseases other than viral infection, were found to be significantly effective in limiting the development of skin lesions and/or in prolonging the mean survival times of HSV-1-infected mice. These results suggested that 12 of 142 HW-extracts that exhibited therapeutic efficacy in an animal infection model were possible candidates for anti-HSV-1 traditional medicine.

Animals↗