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

Rat-2 fibroblasts demonstrate specific binding of I-125-labelled rat adreno medullin (K-D = 0.43 nM; B-max = 50 fmol/mg of protein) in the absence of 1 25I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat- 2 cells were used to examine the pharmacology and signal transduction pathw ays 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 AC1 87 antagonized adrenomedullin-stimulated cAMP production at micromolar conc entrations. Using kinase-substrate assays, Mono Q FPLC and 'phospho-specifi c' Western blotting, we found that adrenomedullin alone abolished basal mit ogen-activated protein kinase (MAPK) activity and dose-dependently inhibite d platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of ad renomedullin-like immunoreactivity of 3.1 fmol/h per 2 x 10(6) cells. Gel-f iltration chromatography showed that this adrenomedullin-like immunoreactiv ity co-eluted with synthetic rat adrenomedullin. Northern blotting with a r at adrenomedullin cDNA probe was used to confirm the presence of adrenomedu llin mRNA. However, neither Northern blotting nor reverse transcriptase-PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclud e that the Rat-2 cell line expresses a specific adrenomedullin receptor (co upled to cAMP production and regulation of MAPK) and secretes adrenomedulli n, which may participate in a regulatory control loop.