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

Abstract

Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD=0.43 nM; Bmax=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 125I-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 2x10(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.

Comparison of the expression of calcitonin receptor‐like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines

“…Rat-2 ®broblasts bound adrenomedullin but not CGRP, as we have previously reported (Coppock et al, 1999). This correlated well with RAMP2 mRNA being present and RAMP1 mRNA being absent.…”

“…High anity binding sites, speci®c for either CGRP or adrenomedullin, have been demonstrated in various tissues and cell lines (Henke et al, 1987;Sexton et al, 1986;Poyner et al, 1992;Owji et al, 1995;Coppock et al, 1999). CGRP receptors have been divided into two subtypes (CGRP 1 and CGRP 2 ) based, predominantly, on the ability of the antagonist CGRP to inhibit the eects of CGRP in functional studies; binding studies are not able to distinguish these putative subtypes (Dennis et al, 1989;1990;Rorabaugh et al, 2001).…”

Vasoactive Hormone Adrenomedullin and Its Binding Protein: Anti-Inflammatory Effects by Up-Regulating Peroxisome Proliferator-Activated Receptor-γ

“…The reason for this is still not completely clear, yet studies have shown that the binding of AM to AMBP-1 prevents its degradation and augments its receptor stimulatory effect (5). The more pronounced reduction of TNF-␣ gene expression compared with the inhibition of the actual release of cytokines by AM/ AMBP-1 indicates that AM/AMBP-1 modulates the proinflammatory response on a transcriptional rather than a secretory level.…”

“…It acts as a circulating or paracrine hormone and mediates its function by binding to a G␣ S protein-coupled calcitonin receptor-like receptor (CRLR), thus activating membrane-bound adenylate cyclases and increasing intracellular cyclic AMP (cAMP) levels (5). Vascular smooth muscle cells consequently react by relaxation, which is both endothelial cell-dependent through the AM-induced release of NO (6) and endothelial cell-independent through a protein kinase A-dependent regulation of calcium dependent K ϩ channels (7).…”

Adrenomedullin in mammalian embryogenesis

“…In a recent report, we have shown that AM binds to human PA-1 teratocarcinoma cell lines and elicits a cAMP response together with an increase in [ 3 H]-thymidine incorporation (Moody et al, 2000). AM has also been found to be expressed and to have specific effects on several lineages of embryonal, fetal, and neonatal origin, such as rat osteoblasts (Cornish et al, 1997), rodent cardiomyocytes (Cormier-Regard et al, 1998;Tsuruda et al, 1998), rat cardiac fibroblasts (Tsuruda et al, 1999), and other embryonic fibroblasts (Coppock et al, 1999;Isumi et al, 1998Isumi et al, , 1999. Interestingly, in some cell types, like mesangial and vascular smooth muscle cells, AM seems to act also as an inhibitor of proliferation, also through a cAMP-mediated signal.…”