Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity

被引：50

作者：

Coppock, HA ^{[1
]}

Owji, AA ^{[1
]}

Austin, C ^{[1
]}

Upton, PD ^{[1
]}

Jackson, ML ^{[1
]}

Gardiner, JV ^{[1
]}

Ghatei, MA ^{[1
]}

Bloom, SR ^{[1
]}

Smith, DM ^{[1
]}

机构：

[1] Univ London Imperial Coll Sci Technol & Med, Hammersmith Hosp, Sch Med, Dept Metab Med,ICSM Endocrine Unit, London W12 0NN, England

来源：

BIOCHEMICAL JOURNAL | 1999年 / 338卷

基金：

英国惠康基金;

关键词：

calcitonin family receptors; signal transduction;

D O I：

10.1042/0264-6021:3380015

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Rat-2 fibroblasts demonstrate specific binding of I-125-labelled rat adrenomedullin (K-D = 0.43 nM; B-max = 50 fmol/mg of protein) in the absence of 125I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat-2 cells were used to examine the pharmacology and signal transduction pathways of adrenomedullin receptors. We examined the effects of adrenomedullin, the CGRP receptor antagonist CGRP-(8-37) and the amylin antagonists AC187 and AC253 on receptor binding and cAMP production. AC253, AC187 and CGRP-(8-37) inhibited I-125-adrenomedullin binding, with respective IC50 values of 25+/-8 129+/-39 and 214+/-56 nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50 = 1.0 nM). CGRP-(8-37), AC253 and AC187 antagonized adrenomedullin-stimulated cAMP production at micromolar concentrations. Using kinase-substrate assays, Mono Q FPLC and 'phospho-specific' Western blotting, we found that adrenomedullin alone abolished basal mitogen-activated protein kinase (MAPK) activity and dose-dependently inhibited platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of adrenomedullin-like immunoreactivity of 3.1 fmol/h per 2 x 10(6) cells. Gel-filtration chromatography showed that this adrenomedullin-like immunoreactivity co-eluted with synthetic rat adrenomedullin. Northern blotting with a rat adrenomedullin cDNA probe was used to confirm the presence of adrenomedullin mRNA. However, neither Northern blotting nor reverse transcriptase-PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclude that the Rat-2 cell line expresses a specific adrenomedullin receptor (coupled to cAMP production and regulation of MAPK) and secretes adrenomedullin, which may participate in a regulatory control loop.

引用

页码：15 / 22

页数：8

共 50 条

[1]

AIYAR N, 1992, MOL CELL BIOCHEM, V113, P17

[2] Proadrenomedullin N-terminal 20 peptide (PAMP) inhibits proliferation of human neuroblastoma TGW cells [J].