Characterization of the adrenomedullin receptor acting as the target of a new radiopharmaceutical biomolecule for lung imaging

Abstract

Direct labeling of linear adrenomedullin (AM) with ^99mTc ([^99mTc]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 [¹²⁵I]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 ≈ AM2 ≥ AM(22–52) ≥ AM2(16–47) > CGRP(8–37) > amylin ≈ 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 AM₁ receptor subtype. The pharmacophores underlying AM₁ 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 α-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.

Introduction

The multigenic calcitonin family is composed of calcitonin, amylin, two calcitonin-gene related peptides (CGRPα and CGRPβ), adrenomedullin (AM), intermedin or AM2 and the newly discovered calcitonin receptor stimulating peptides (CRSP) (Table 1) (Katafuchi et al., 2003, Roh et al., 2004, Takei et al., 2004). Adrenomedullin (AM) was originally isolated from human pheochromocytoma and characterized by its ability to raise intracellular cAMP levels in rat platelet (Kitamura et al., 1993). AM, which possesses 25% homology with CGRPα and 28% with AM2, shares partial structural homology with calcitonin family peptides depicted by a six amino acid ring and, except for the CRSP, a C-terminal amide function well conserved among vertebrates (Bell and McDermott, 2008, Hinson et al., 2000, Poyner et al., 2002).

Numerous studies demonstrated that AM exerts pleiotropic actions (Hinson et al., 2000) including cell proliferation (Shichiri and Hirata, 2003), migration (Fukai et al., 2003), apoptosis (Shichiri et al., 1999), inflammation (Hirata et al., 1996, Sugo et al., 1995), angiogenesis (Kim et al., 2003), and hormone secretion (Nikitenko et al., 2002). However, the major role of AM, which is a paracrine control of vascular function (particularly microvascular), is supported by the high levels of peptide secreted by endothelial and vascular smooth muscle cells (Chu et al., 2001).

Peptides from the calcitonin family mediate their action through different heterodimer complexes between a conventional GPCR [known as calcitonin receptor (CTR) or calcitonin receptor-like receptor (CLR)] and a single transmembrane protein called receptor activity-modifying protein (RAMP) (Parameswaran and Spielman, 2006). Pharmacological studies revealed that at least three peptides, namely CGRP, amylin, and AM2, are able to bind AM receptors that are formed by the co-expression of RAMP2 or RAMP3 with CLR (Born et al., 2002, McLatchie et al., 1998, Parameswaran and Spielman, 2006, Poyner et al., 2002).

Earlier studies have demonstrated that AM is secreted from various tissues, including vessels, heart, and lungs (Ichiki et al., 1994, Sakata et al., 1994). Important specific AM binding sites were reported in rat and human lungs and it was shown that these receptors exhibit different pharmacological properties than receptors found in the heart (Martinez et al., 1997, Owji et al., 1995).

Therefore, due to the high density of AM receptors in the cardio-respiratory system and the fact that AM metabolism occurs primarily via receptor binding in a variety of tissues including lungs (Dupuis et al., 2005), it was proposed that AM receptors could represent a key-target useful for the diagnosis of disorders of the pulmonary circulation with radiolabeled AM-related drugs. Recently, we reported the use of [^99mTc]AM to obtain highly selective images of the pulmonary circulation (Fig. 1; Harel et al., 2008). Moreover, pharmacokinetics and pharmacodynamics of [^99mTc]AM, as evaluated in dogs, supported the pulmonary specificity and clearance of AM by lungs.

This study aimed at identifying the pharmacological profile of [^99mTc]AM receptor-specific binding sites found in dog lung. Using known agonists and antagonists of the calcitonin peptide family and [¹²⁵I]AM used as the radioligand, we were able to demonstrate that dog lung homogenates express a specific AM receptor that exhibits AM₁ properties.