Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD = 0.43nM; Bmax = 50fmol/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 125I-adrenomedullin binding, with respective IC50 values of 25±8, 129±39 and 214±56nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50 = 1.0nM). 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 3.1fmol/h per 2×106 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.
Present address: Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran.
Present address: Secretory Pathway Laboratory, ICRF, Lincoln's Inn Fields, London WC2A 3PX, U.K.