Isolation and cloning of VIP receptors.
| Project/Area Number |
03454101
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
基礎獣医学
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| Research Institution | Hokkaido University |
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Principal Investigator |
ITO Shigeo Hokkaido University, Faculty of Veterinary Medicine, Department of Pharmacology, Associate Professor., 獣医学部, 助教授 (40109509)
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| Co-Investigator(Kenkyū-buntansha) |
OHTA Toshio Hokkaido University, Faculty of Veterinary Medicine, Department of Pharmacology,, 獣医学部, 助手 (20176895)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1992: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1991: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | vasoactive intestinal peptide / canine liver / VIP binding experiment / PACAP / VIP receptors / cloning / vascactive imtestinal peptide / パラサイロイドホルモン受容体 / Vasoactive intestinal peptide / VIP受溶体 / イヌ肝臓細胞膜 / PCR法 |
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| Research Abstract |
The binding characteristic of vasoactive intestinal peptide (VIP) receptors was examined using radioligand binding assay with iodinated VIP and unlabelled peptides in liver mambranes of the dog. VIP binding to canine liver membranes was rapid, reversible, saturable, specific and dependent on temperature. Guanine nucleotides dose-dependently inhibited VIP binding. Stoichiometric data suggested the presence of two classes of VIP binding sites. The order of potency by competition experiments with unlabelled peptides was: VIP > pituitary adenylate cyclase activating peptide (PACAP)-27 > PACAP-38 >> peptide histidine isoleucine (PHI) = secretin in the dog and PACAP-27 > PACAP-38 > VIP > PHI > secretin in the rat. PHI and secretin were about 5000 times less potent than VIP in the dog but about 100 times less potent in the rat. It was concluded that the VIP receptors in canine liver membranes possesses recognition sites for VIP which were distinct from those in rat liver membranes. To obtain DNA probe for screening. VIP receptor gene of the rat liver was amplified by polymerase chain reaction using synthesized oligonucleotide primers. The products, 540-base-pair fragments, were purified and served as the probe after labelling. A cDNA library (300000 clones) derived from canine liver was screened under low stringency conditions. Six positive colonies were detected. Four out of 6 clones with larger inserts (1-1.5 kbb) were subjected to nucleotide sequence analysis. #35 clone has a nucleic acid sequence identical to parathyroid hormone receptors of the rat and opossum. #102 clone has a nucleic acid sequence similar to rat VIP receptors but not #201 and #513 clones. The analysis of #102 clone is in progress.
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Report
(3 results)
Research Products
(6 results)
