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F Ushikubi

Publications and source records attributed to F Ushikubi.

79 records · Page 5Linked to original sources
PubMed1986

Binding of a radioiodinated 13-azapinane thromboxane antagonist to platelets: correlation with antiaggregatory activity in different species.

Binding of a 125I-labelled derivative of the 13-azapinane thromboxane antagonist (ONO-11120), [125I]-9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyp hen yl)-13, 14-dihydro-13-aza-15-beta-omega-tetranor-thromboxane A2 ([125I]-PTA-OH), to washed platelets of human, dog and rabbit was studied. Results were compared with the in vitro inhibitory potency of ONO-11120 on platelet aggregation induced by arachidonate and a thromboxane agonist, 9,11-epithio-11,12-methano-thromboxane A2 (STA2). [125I]-PTA-OH bound to washed human platelets in a reversible, saturable and temperature-dependent manner, and specific binding displaced by 20 microM ONO-11120 constituted about 40% of the total binding. Scatchard analyses revealed a single class of specific binding and the equilibrium dissociation constant (KD) and maximal concentration of binding sites (Bmax) were 22 nM and 390 fmol per 10(8) platelets (about 2,300 sites per platelet), respectively. In addition to ONO-11120, STA2 and another thromboxane receptor agonist, (15S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid (U-46619), effectively displaced the binding with IC50 values of 44 and 125 nM respectively. Prostaglandin D2 (PGD2) partially displaced the binding only at a concentration above 1 microM. PGE1 and thromboxane B2 (TXB2) were without effect up to 100 microM. Similar binding of [125I]-PTA-OH was observed on dog platelets. The KD and Bmax were 12 nM and 110 fmol per 10(8) platelets (about 680 sites per platelet), respectively, and these values did not change significantly after adrenaline treatment which potentiated arachidonate-induced aggregation of platelets in this species. On the other hand, no specific binding of ['251]-PTA-OH was found on rabbit platelets. 4 Consistent with the results from binding studies, ONO-11120, 0.5 microM, completely suppressed arachidonate-induced aggregation of human platelets, whereas, at concentrations up to 5 microM, this agent did not significantly inhibit aggregation of rabbit platelets induced by the same stimulus. STA2- induced aggregation of rabbit platelets also showed less sensitivity to ONO-I 1120. When a similar extent ofirreversible aggregation was induced by STA2 and the inhibitory potency ofONO-1 1120 was compared in human and rabbit platelets, about one hundred times greater concentration of ONO- 11120 was required to suppress aggregation of rabbit platelets than that of human platelets. 5 These results suggest that [1251]-PTA-OH binds to a platelet thromboxane receptor, and that the structure of the binding site(s) on the receptor may vary between species.

Animals↗
PubMedDate not supplied

Structure and function of prostanoid receptors.

We have recently cloned cDNAs for the human and mouse TXA2/PGH2 receptors and a cDNA for the mouse PGE receptor. Sequencing, homology and hydrophobicity analyses revealed that they are proteins of 343, 341 and 365 amino acid residues, respectively, and all are rhodopsin-type receptors with putative seven transmembrane domains. Homology between the human and mouse TXA2 receptors is 76% in total and that between the human TXA2 and mouse PGE receptors is 38%. The homology increases in the putative transmembrane regions to 85 and 45%, respectively, and there observed several features common to the three receptors. These results indicate that the prostanoid receptors constitute a family of receptors of similar structure. The cloned PGE receptor showed binding activity specific to so-called EP3 agonists, which verified for the first time that pharmacologically defined PGE receptor subtypes consist of different molecules. This receptor also displayed different efficiency in signal transduction to an EP3 agonist, M & B-28767, and PGE2, providing an interesting model for the analysis of receptor-G protein coupling. In addition to the above biochemical results, these studies have revealed the characteristic tissue distribution of these receptors, which will open up a new biology of these prostanoids.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular characterization of a dominantly inherited bleeding disorder with impaired platelet responses to thromboxane A2.

Thromboxane A2 (TXA2) is a major arachidonic acid metabolite of platelets and induces platelet functions by binding to specific receptors on the membrane. We have found patients with hemostatic defects due to impaired platelet responses to TXA2, and molecular characterization of the patients has been carried out. Platelets from these two unrelated patients showed impaired aggregation responses to TXA2 and its analogues despite the normal response to thrombin. Although the patients' platelets exhibited normal binding activities to TXA2 analogues, they showed decreased GTPase activity and second messenger formation when stimulated by STA2, a stable TXA2 agonist. To understand the molecular basis of this abnormality, we determined the cDNA sequence of the TXA2 receptor by reverse transcription-polymerase chain reaction (RT-PCR) from the patient's platelet RNA, and identified a single amino acid substitution (Arg60 for Leu) in the first cytoplasmic loop of the receptor. This mutation was found in both isoforms of the platelet TXA2 receptor which we have recently found: TXR alpha with the same structure as the placental TXA2 receptor and TXR beta with the same structure as the endothelial TXA2 receptor, and was detected exclusively in affected members of two unrelated families with the disorder. The mutant TXR alpha and TXR beta expressed in COS-m6 cells showed decreased agonist-induced phospholipase C activation despite their normal ligand binding affinities. These results suggest that the Arg60 for Leu mutation is responsible for the disorder and imply a critical role for the first cytoplasmic loop in the interaction of the TXA2 receptor with the G protein.

Base Sequence↗