Adrenomedullin receptor binding sites in rat brain and peripheral tissues

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Abstract

The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13–52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13–52) binds to an apparent single population of sites with similar affinities (KD of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (Bmax of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1–52) and human adrenomedullin-(13–52) competed against specific [125I]human adrenomedullin-(13–52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide α (hCGRPα), [Cys(Et)2,7]hCGRPα and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13–52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPγS (100 μM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13–52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1–52) and [Cys(Et)2,7]hCGRPα against specific [125I]human adrenomedullin-(13–52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13–52) binding sites that are resistant to human adrenomedullin-(22–52) and human CGRP-(8–37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.

Introduction

Human adrenomedullin is a 52-amino acid peptide isolated from human adrenal pheochromocytoma (Kitamura et al., 1993). Both human and rat adrenomedullin are derived from a 185-amino acid preproadrenomedullin; however, the mature form of rat adrenomedullin is a 50-amino acid peptide (Sakata et al., 1993).

Adrenomedullin-like immunoreactivity and its corresponding mRNA have been reported to be expressed in various tissues including the adrenal gland, heart, lungs and kidneys Ichiki et al., 1995, Kitamura et al., 1994, Sakata et al., 1994 as well as in the hypothalami of the human and rat Satoh et al., 1996, Ueta et al., 1995. As reported for calcitonin gene-related peptide (CGRP) and to a lesser extent amylin, intracerebroventricular injections of adrenomedullin increased blood pressure (Takahashi et al., 1994) inhibited water intake (Murphy and Samson, 1995) and produced anorexia (Taylor et al., 1996), while inducing a potent vasodilatation in peripheral tissues (Nuki et al., 1993). On the basis of the comparative biological effects induced by adrenomedullin and CGRP, early studies suggested that both peptides were acting via a common CGRP-like receptor Eguchi et al., 1994a, Taylor et al., 1996. For example, it has been shown that the potent vasodilator action of adrenomedullin was antagonized by a CGRP receptor antagonist, CGRP-(8–37), suggesting the involvement of the CGRP1-like receptor subtype Baskaya et al., 1995, Berthiaume et al., 1995, Eguchi et al., 1994b, Entzeroth et al., 1995, Hall et al., 1995. Similarly, the anorexic property and hypertensive effects of brain administered adrenomedullin were proposed to be mediated via CGRP1 receptors (Taylor et al., 1996).

However, various in vitro and in vivo bioassays studies supported the existence of a specific population of adrenomedullin receptors on the basis of the very low to low potencies of CGRP-(8–37) to antagonize the effects of adrenomedullin in rat systemic vascular bed (Nandha et al., 1996), guinea pig pulmonary artery (Pinto et al., 1996), mouse astrocytes or renal glomeruli Hjelmqvist et al., 1997, Yeung et al., 1996 and aldosterone secretion Hinson et al., 1998, Kapas et al., 1998. Additionally, receptor binding assays have shown that hCGRPα is 100 to 1000 times less potent than adrenomedullin to compete for specific [125I]human or rat adrenomedullin-binding sites in the rat spinal cord (Owji et al., 1995), rat systemic vascular bed (Nandha et al., 1996) and in a rat skeletal muscle cell line (L6) (Coppock et al., 1996) as well as in several other tissues (Hinson et al., 2000). Moreover, structure–activity studies revealed that removing the first 21 N-terminal amino acid residues of human adrenomedullin, just next to the disulfide bridge (human adrenomedullin-(22–52)), generated a fragment with antagonistic properties (Coppock et al., 1996). In most cases, human adrenomedullin-(22–52) was able to block the effects of adrenomedullin without affecting those of CGRP Fujioka et al., 1999, Gardiner et al., 1999, Nishikimi et al., 1998. Taken together, these results suggested that at least some of biological effects of adrenomedullin were mediated by specific adrenomedullin receptors.

One of the most convincing evidence for the existence of a distinct adrenomedullin receptors was provided by the cloning of single transmembrane proteins known as the receptor-activity-modifying proteins (RAMP) which confer to the calcitonin-receptor like receptor (CRL receptor) either a CGRP or an adrenomedullin-like ligand selectivity profile depending on the associated RAMP (Poyner et al., 2002). When CRL receptor is co-transfected with either RAMP2 or RAMP3, an adrenomedullin receptor was generated while the co-transfection of CRL receptor with RAMP1 resulted in a functional CGRP receptor in human embryonic kidney (HEK) 293 cells (McLatchie et al., 1998).

Good correlation between the distribution of CGRP binding sites and CRL receptor/RAMP1, as well as adrenomedullin receptors and CRL receptor/RAMP2 has been shown in several tissues (Chakravarty et al., 2000), However, this may not account for the totality of [125I]adrenomedullin and [125I]CGRP receptor-binding sites. For example, the expression of the CRL receptor mRNA failed to be detected in the spinal cord and cerebellum Fluhmann et al., 1997, Oliver et al., 1998, two areas highly enriched with specific [125I]adrenomedullin and [125I]hCGRPα-binding sites Jacques et al., 2000, Juaneda et al., 2000, Juaneda et al., 2001, Owji et al., 1995, Van Rossum et al., 1997. Moreover, in rat aortic vascular smooth muscle cells, a prototypical adrenomedullin bioassay, CRL receptor mRNA is not expressed (Autelitano and Tang, 1999), suggesting the possible existence of other genes coding for additional adrenomedullin receptors.

We studied here the respective binding profile of [125I]human adrenomedullin-(13–52) (a potent agonist) in rat brain, lung and vas deferens membrane homogenates. Our results suggest that [125I]human adrenomedullin-(13–52) may recognize heterogeneous populations of receptor-binding sites in these tissues.

Section snippets

Materials

Male Sprague–Dawley rats (250–300 g) were obtained from Charles River (St-Constant, QC, Canada) and kept on a 12-h light, 12-h dark cycle (lights on at 07:00) in temperature- and humidity-controlled rooms. Animals were fed with standard laboratory chow and had access to tap water ad libitum. Animal care was according to the protocols and guidelines approved by McGill University and the Canadian Council of Animal Care.

Human calcitonin gene-related peptide alpha (hCGRPα), hCGRP-(8–37), [Cys(ACM)

Results

Prototypical isotherm saturation binding experiments revealed that [125I]human adrenomedullin-(13–52) binds to an apparent single population of sites in the rat lung (Fig. 1) as well as in rat brain and vas deferens (data not shown). Binding parameters derived from saturation-binding experiments demonstrate that [125I]human adrenomedullin-(13–52) binds with high affinity (KD=0.32±0.05, 0. 66±0.16 and 0.33±0.11 nM) and saturable amount (Bmax=73±4, 1760±170 and 144±10) of sites in rat brain, lung

Discussion

This study provides evidence for the existence of the possible heterogeneity of adrenomedullin receptors in rat tissues. This hypothesis is based on the differential affinity and ability of some competitors such as human adrenomedullin-(22–52), hCGRPα, hCGRP-(8–37), [Cys(Et)2,7]hCGRPα and BIBN4096BS to inhibit only a fraction of specific [125I]human adrenomedullin-(13–52) binding sites. Furthermore, the existence of an heterogeneous population of [125I]human adrenomedullin-(13–52) sites in the

Acknowledgments

This study was supported by grants from the Canadian Institutes of Health Research (CIHR) to R. Quirion. A. Fournier is a “Chercheur-Boursier” of the “Fonds de la Recherche en Santé du Québec (FRSQ)”.

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