1. Characterization of the adrenomedullin receptor acting as the target of a new radiopharmaceutical biomolecule for lung imaging
Alain Fournier, Yan Fu, Quang T Nguyen, David Chatenet, Myriam Létourneau, Jocelyn Dupuis Eur J Pharmacol . 2009 Sep 1;617(1-3):118-23. doi: 10.1016/j.ejphar.2009.06.031.
Direct labeling of linear adrenomedullin (AM) with (99m)Tc ([(99m)Tc]AM) displayed excellent selectivity for imaging the pulmonary circulation system in dogs. Hence, we investigated this particular selectivity and characterized the binding sites found in dog lungs. AM and other peptides belonging to the calcitonin peptide family, including calcitonin-gene related peptide (CGRP), adrenomedullin-2 (AM2), amylin and pro-adrenomedullin N-terminal peptide (PAMP), were prepared by solid-phase peptide synthesis. Receptor binding assays were performed by using [(125)I]AM as a radioligand on dog lung homogenates. It was found that AM bound with potent affinity, displaying in fact a high and a low affinity binding site. Moreover, competition binding assays using peptide ligands showed the following ranking for displacement: AM>AM(13-52)>CGRP approximately AM2> or =AM(22-52)> or =AM2(16-47)>CGRP(8-37)>amylin approximately PAMP. Thus, these results strongly suggested that the AM binding site found in dog lungs and acting as a clearance receptor is mainly the adrenomedullin AM(1) receptor subtype. The pharmacophores underlying AM(1) binding affinity and specificity were studied by determining the key amino acids, the minimal peptide fragment, and some aspects of the secondary structures. So far, it appeared that the C-terminal segment of human AM is an essential feature for binding. Also, the alpha-helix secondary structure found in the AM molecule would facilitate the ligand recognition process with the AM receptor in dog lungs. Our results demonstrated that AM or some analogs or fragments could be suitable radiopharmaceutical agents for lung imaging.
2. Adrenomedullin(1-52) measured in human plasma by radioimmunoassay: plasma concentration, adsorption, and storage
T G Yandle, L K Lewis, M W Smith, M G Nicholls, A M Richards Clin Chem . 1998 Mar;44(3):571-7.
We describe a specific and sensitive RIA for human adrenomedullin (AM)(1-52). The detection limit and the concentration required for 50% inhibition of binding were 0.1 and 1.2 fmol/tube, respectively. Cross-reactivities with AM(1-12), AM(13-52), calcitonin gene-related peptide, amylin, and other vasoactive hormones were negligible. AM immunoreactivity in normal subjects ranged from 2.7 to 10.1 pmol/L (n = 44). We investigated factors influencing the recovery and measurement of AM in the assay. Recovery of labeled AM (> 80%) was markedly higher than that of unlabeled AM (56%). Immunoreactivity of exogenous AM added to plasma decreased up to 70% over four freeze-thaw cycles, whereas endogenous AM was stable. Alkali-treated casein (1 g/L) reduced adsorption of AM to surfaces and significantly increased assay precision compared with bovine serum albumin (P < 0.0001). HPLC separation of extracted plasma verified the presence of AM(1-52). We suggest that considerable care is needed to ensure that accurate and reproducible results are obtained from studies quantifying this peptide.
3. Analysis of responses to adrenomedullin-(13-52) in the pulmonary vascular bed of rats
H C Champion, Q Hao, P J Kadowitz, A L Hyman, B Gumusel, H Lippton, J L Mehta, J K Chang Am J Physiol . 1998 Apr;274(4):H1255-63. doi: 10.1152/ajpheart.1998.274.4.H1255.
The effects of human adrenomedullin-(13-52) [hADM-(13-52)] were investigated in the rat pulmonary vascular bed and in isolated rings from the rat pulmonary artery (PA). Under conditions of controlled blood flow and constant left atrial pressure when tone was increased with U-46619, injection of hADM-(13-52) produced dose-related decreases in lobar arterial pressure. Pulmonary vasodilator responses in the intact rat and vasorelaxant responses to hADM-(13-52) in rat PA rings were inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and L-N5-(1-iminoethyl)-ornithine hydrochloride (L-NIO). Vasorelaxant responses to hADM-(13-52) were also inhibited by methylene blue, endothelium removal, hADM-(26-52), and iberiotoxin, whereas meclofenamate, calcitonin gene-related peptide-(8-37) [CGRP-(8-37)], glibenclamide, and apamin were without effect. Because vasorelaxant responses to NS-1619, a large-conductance Ca(2+)-activated K+ channel agonist, were not altered by L-NAME and vasorelaxant responses to acetylcholine and CGRP were not altered by hADM-(26-52), the present data suggest that ADM-(13-52) acts on a receptor in the pulmonary vascular bed that is coupled to endothelial nitric oxide release. These data suggest that this nitric oxide release may lead to guanosine 3',5'-cyclic monophosphate-dependent K+ channel activation, which produces a pulmonary vasorelaxant response through hyperpolarization of vascular smooth muscle cells. The present data suggest that ADM-(13-52) modulates receptor-mediated, but not voltage-dependent, pulmonary vascular contraction by influencing Ca2+ influx. These results suggest that the ADM fragment, hADM-(13-52), acts as an endothelium-dependent vasodilator agent in the pulmonary vascular bed of the rat.
