Atsushi Irie

Specific binding of glycyrrhetinic acid to the rat liver membrane.

Glycyrrhetinic acid bound specifically to a particulate fraction of rat liver. The binding was dependent on time, temperature and pH, equilibrium being reached after 10 min at 37 degrees C. The equilibrium dissociation constant and the maximal concentration of the binding site, as determined by Scatchard plot analysis, were 31 nM and 43 pmol/mg protein, respectively, indicating a single binding site entity. The binding site was highly specific for glycyrrhetinic acid, glycyrrhizin, various steroids, various fatty acids and retinoids showing no or only very low affinity. The binding was inhibited by boiling or treatment with trypsin or phospholipases. The specific activity of glycyrrhetinic acid binding was the highest in the liver, followed by in the kidney. The results suggest that glycyrrhetinic acid plays a significant role in the rat liver through its specific binding protein.

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 PGF2α 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.

Induction of A 31,000-dalton stress protein by prostaglandins D2 and J2 in porcine aortic endothelial cells

Prostaglandin (PG) D2 and PGJ2 stimulated porcine aortic endothelial cells to synthesize a 31,000-dalton protein (termed p31) in a time- and concentration-dependent manner. The induction of p31 synthesis was specific for PGD2, PGJ2 and PGA1 among the various PGs tested. p31 was also synthesized in response to the thiol-reactive agent diethylmaleate and heavy metal sodium arsenite but not to high temperature treatment, platelet-derived growth factor, and 12-O-tetradecanoylphorbol 13-acetate. Using two-dimensional polyacrylamide gel electrophoresis, p31 induced by PGJ2 had an isoelectric point of 5.4, which overlapped exactly with that induced by by arsenite. These results taken together indicate that p31 represents one of the stress proteins whose expression is regulated primarily by thio-active compounds but not by hyperthermia. Furthermore, it was induced by PGD2 and PGJ2 in rat capillary endothelial cells, rat skin fibroblasts, and rat hepatocytes. The data obtained from this study suggest that p31 induced by PGD2 and PGJ2 may play a role in the metabolic regulation of many mammalian cells.

Glycyrrhetinic acid bound to 11β-hydroxysteriod dehydrogenase in rat liver microsomes

Abstract A binding protein which exibits high affinity to [ 3 H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [ 3 H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11β-hydroxysteriod dehydrogenase (11β-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11β-HSD activity. These results show that the purified [ 3 H]glycyrrhetinic-acid binding protein is 11β-HSD. From the molecular mass of 11β-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11β-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [ 3 H]glycyrrhetinic acid binding and 11β-HSD activity indicate that the C 30 -carboxyl and C 11 -carbonyl groups of glycyrrhetinic acid are the principal structures for the 11β-HSD inhibition.