Biochemical Characterization and Cloning of Prostaglanoin D_2 Recoptor
| Project/Area Number |
02670118
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Osaka Bioscience Institute |
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Principal Investigator |
ITO Seiji Osaka Bioscience Institute, 4th Dept., Subhead, 第4研究部, 副部長 (80201325)
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| Co-Investigator(Kenkyū-buntansha) |
芦高 恵美子 大阪バイオサイエンス研究所, 第4研究部, 研究助手
ASHITAKA Emiko (OKUDA Em) Osaka Bioscience Institute, 4th Dept., Technician
小田 紀子 大阪バイオサイエンス研究所, 第4研究部, 共同研究員
洲鎌 和茂 大阪バイオサイエンス研究所, 第4研究部, 研究員 (40206397)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1991: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Prostaglandin D_2 / cAMP / onchromaffin cells / Receptor / Cloning / FRTL5 cells / EBTr cells / Astrocytes / プロスタグランジンD_2(PGD_2) / アデニレ-トシクラ-ゼ |
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| Research Abstract |
Although many biological actions of prostaglandin (PG) D_2 such as sleep induction and inhibition of platelet aggregatio, have been reported, little information is currently available concerning the mode of coupling of PGD_2 receptors to Ca^<2+> mobilization and the molecular structure of the receptor. This is mainly due to failure in detection of PGD_2 binding activity in tissues other than and platelets and the concentration of the receptor in tissues being very limited. Recently we found that nonchromaffin cells of bovine adrenal medulla and a cell line derived from bovine embryonic tracheal (EBTr) cells elevated cAMP level specifically by PGD_2. In this study, we tried to clone cDNA encoding PGD_2 receptors (I), characterized mechanisms linked to Ca^<2+> mobilization using cuitured cells (II), and reported new biological effects of PGD_2 (III).
(I) A cDNA library of nonchromaffin cells was generated in the expression vector pcD2 according to the method of Okayama-Berg. After the siz
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e fractionation of cDNAs by the agarose gel electrophoresis, large size cDNA population (<greater than or equal> kb) was recloned in the same plasmid vector and transfected into FRTL5 cells by the Ca^<2+>-phosphate precipitation method. About 1500 stable transformants were screened by G418 selection and a clone A4-7 which reproducibly elevated the cAMP level 1.4-1.8-fold by PGD_2 was finally obtained. This elevation was blocked by PGD_2 antagonists BWA868C and AH6809, suggesting that this clone may express the PGD_2 receptor on the cell. Plasmid DNAs containing 15 different inserts were recovered by the Hirt method. Subclones of A4-7 were transfected into FRTL5 cells and screened for cAMP elevation by PGD_2. A PGD_2-resnsive clone A4-7-30 with a 1.3 kb insert was obtained and sequence analysis of this clone is in progress.
(II) We demonstrate that there are two mechanisms of Ca^<2+> mobilization by PGD_2. In EBTr cells, PGD_2 increases intracellular Ca^<2+> concentration ([Ca^<2+>]_i) mediated by cAMP through PGD_2 receptor. In rat astrocytes, PGD_2 increases [Ca^<2+>]_i by stimulating phosphoinositide metabolism through the PGF_<2alpha>/PGD_2 receptor. These two signal transduction pathways may account for apparent subrypes of PGD_2 receptors among PGD_2-sensitive tissues and cells reported previously.
(III) Although the in vivo and in vitro inhibitory effects of PGD_2 on cell growth have been attributed to the dehydrate product DELTA^<12>-PGJ_2, this study is the first to demonstrate that PGD_2 exerts an inhibitory effect on cell growth and c-myc expression in EBTr cells via cAMP. Intrathecal administration of PGD_2 and PGF_<2alpha> into mice is shown to evoke allodynia, a state of discomfort and pain induced by innocuous stimuli. This is the first exarnple of allodynia induced by natural substances and could be a model of patients with chronic pain. Less
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Report
(3 results)
Research Products
(17 results)
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[Publications] Ito, S., Okuda, E., Negishi, M., Sugama, K., and Hayaishi, O.: "Evaluation of ZK110841 and AH6809, an agonist and an antagonist of prostaglandin DP-receptors on human platelets, with a PGD_2 -responsive cell line from bovine embryonic trachea." Br. J. Pharmacol.99. 13-14 (1990)
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[Publications] Oda, K., Fujitani, Y., Watanabe, T., Inui, T., Okada, T., Urade, Y., Okuda-Ashitaka, E., and Ito, S.: "Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells." FEBS Lett. 299. 187-191 (1992)
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[Publications] Ito, S., Sugama, K., Inagaki, N., Fukui, H., Giles, H., Wada, H., and Hayaishi, O.: "Type-1 and type-2 astrocytes are distinct tergets for prostaglandin D_2, E_2, and F_2alpha." Glia. (1992)
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[Publications] Minami, T., Uda, R., Horiguchi, S., Ito, S., Hyodo, M., and Hayaishi, O.: "Allodynia evoked by intrathecal administration of prostaglandin F_2alpha to conscious mice." Pain. (1992)
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