Elsevier

Brain Research

Volume 997, Issue 1, 30 January 2004, Pages 97-102
Brain Research

Research report
Peripheral amylin activates circumventricular organs expressing calcitonin receptor a/b subtypes and receptor-activity modifying proteins in the rat

https://doi.org/10.1016/j.brainres.2003.10.040Get rights and content

Abstract

The pancreatic hormone amylin (AMY) and the AMY-receptor-agonist salmon-calcitonin (sCT) reduce short-term food-intake after binding to the area postrema (AP), a circumventricular organ (CVO) lacking blood–brain-barrier characteristics. AMY has also been proposed to induce drinking via another CVO, the subfornical organ (SFO). In cellular systems, AMY-binding is generated by interaction of calcitonin-receptor a/b (CT(a)/CT(b)) with receptor-activity modifying proteins (RAMPs). By using in situ hybridization, the codistribution of CT(a)/CT(b) with RAMP1–3 and c-fos was mapped in CVOs of rats. AMY and sCT induced c-fos within the SFO which contained CT(a) and/or CT(b) and RAMP1/2 mRNA. AMY and sCT also activated AP neurons, which express the CT(a), but not the CT(b), receptor and RAMP2/3 mRNA. These data emphasize the important role of these structures as primary targets for circulating AMY.

Introduction

The 37-amino acid peptide hormone amylin (AMY) belongs to the family of calcitonin gene-related peptides and is co-secreted with insulin by pancreatic β-cells in response to feeding. Acute peripheral application of AMY suppresses feeding in rats and mice [7] through a central mechanism [8], [10].

As analyzed by in vitro autoradiography, binding sites for [125I] rat AMY were found in various hypothalamic nuclei [20], [23]. Furthermore, strong labelling of [125I] rat AMY is present in the subfornical organ (SFO) and the area postrema (AP) [20], which represent sensory circumventricular organs (CVOs) lacking a functional blood–brain barrier.

AMY binding sites belong to the class of calcitonin (CT) receptors. In cellular systems high affinity AMY binding requires the coexpression of the CT receptor with distinct receptor-activity modifying proteins (RAMPs). These RAMPs comprise a family of three accessory single transmembrane proteins (designated RAMP1, RAMP2 and RAMP3), which were shown to heterodimerize with the CT receptor or with the calcitonin receptor-like receptor (CL receptor) to define the distinct ligand specificity of these G protein-coupled receptors [5], [12], [13], [16], [22]. By using co-transfection strategies, RAMP1 and RAMP3 transfected into CT receptor expressing cells generate an AMY receptor phenotype, while the potential of RAMP2/CT receptor heterodimer to induce AMY binding was highly dependent upon the cellular background [22]. Ligand binding of CT or the AMY receptor agonist salmon CT (sCT) to the CT receptor did not depend on RAMPs heterodimers with the CT receptor [5].

Beside the CT receptor, RAMP interaction is further shown for the human CL receptor isoform to determine the specific binding of adrenomedullin or CGRP. CL receptor heterodimers with RAMP1 correspond to the native CGRP receptor, whereas association of CL receptor with RAMP2 or RAMP3 bind adrenomedullin with high affinity. Although heterodimerization of RAMPs determines ligand specificity of both CT and CL receptors, the RAMP dependent translocation of receptor molecules to the cell surface seems to be restricted to the CL receptor while CT receptor expression at the cell surface is independent of the presence of RAMPs [16].

Cloning studies demonstrated two calcitonin receptor subtypes in rodents, termed CT(a) and CT(b) receptor isoforms, which are identical, except that the CT(b) sequence contains an additional 37-amino acid insert in the first extracellular loop which is associated with a seven- to eight-fold lower binding affinity for the AMY receptor agonist sCT [1]. Regarding the potential to generate AMY binding sites, both CT(a) and CT(b) isoforms of the CT receptor can be modified by RAMP1 and RAMP3 to function as an AMY receptor [16].

Several functional studies have demonstrated that the AP and the SFO are the main central targets which mediate AMY's anorectic effect and its suggested drinking response, respectively [10], [17]. In addition, both sensory CVOs are activated by AMY [17], [18] and were shown to contain high affinity AMY binding sites [20]. However, in situ hybridization studies mapping the distribution of CT receptor mRNA [21] and RAMP1–3 mRNA [14] in the rat brain have not described the respective expression profiles in these circumventricular structures.

It was therefore the aim of the present study to provide detailed information about the expression pattern of these molecular components necessary for AMY binding focussing on the SFO and AP as sensory CVOs.

Section snippets

Animals

Adult male Wistar rats (BRL, Füllinsdorf, Switzerland; mean body weight 240 g) used for the experiments were housed individually and adapted to the housing conditions (room temperature 21±1 °C, artificial 12-h/12-h light/dark cycle) for at least 2 weeks before the experiments. Medium fat diet (18% w/w fat) and water was available ad libitum. Rats (n=4 per group) were injected intraperitoneally (i.p.) at dark onset with either AMY (50 μg/kg BW), the AMY receptor agonist sCT (50 μg/kg BW) or

Food intake

The 60- and 120-min cumulative food intake significantly decreased after i.p. injection of AMY (50 μg/kg BW) and was almost abolished after application of sCT (50 μg/kg BW) when compared to the NaCl-treated control group (60 min: NaCl 1.63±0.38 g, AMY 0.65±0.09 g*, sCT 0.02±0.03 g**; 120 min: NaCl 2.75±0.64 g, AMY 1.10±0.28 g*, sCT 0.05±0.05 g**; *p<0.05, **p<0.01).

In situ hybridization

Non-specific hybridization was carried out in the presence of excess unlabelled antisense ODNs hybridized to alternate tissue

Discussion

Recently, we demonstrated that acute application of AMY and sCT induced short term anorexia and modulated the gene expression of orexigenic neuropeptides within the lateral hypothalamic area [2]. The anorectic action of AMY and sCT is not mediated by vagal or non-vagal afferents [8] but by AMY specific binding sites in the central nervous system [3]. While the lateral hypothalamic area is located inside the blood–brain barrier without direct access to peripheral circulation, the AP as sensory

Acknowledgements

The authors are grateful to the technical assistance of Doris Haase. This study was financially supported by the Swiss National Research Foundation No. 31-66694.01.

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