Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat endothelium

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Abstract

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.

Introduction

Calcitonin gene-related peptide (CGRP) and adrenomedullin are structurally homologous peptides (Poyner et al., 2002) that exert similar biological effects. Both peptides have potent vasodilatory actions that appear to be mediated by a common G protein-associated receptor, the calcitonin receptor-like (CRL) receptor (Chu et al., 2001). CRL receptor has been cloned from several species Born et al., 2002, Poyner et al., 2002 and acts either as a CGRP or an adrenomedullin receptor, depending with which receptor activity-modifying proteins (RAMPs) it is co-expressed (McLatchie et al., 1998). The CRL receptor/RAMP1 complex is the pharmacologically defined CGRP-1 receptor with which both CGRP and adrenomedullin interact, while RAMP2 or RAMP3 in combination with CRL receptor is an adrenomedullin receptor. The precise cellular localisation of CRL receptor in the rat is not yet determined due to the lack of an available and well-characterised antibody for precise immunochemical analysis. We aimed to develop and fully characterise an antibody for rat CRL receptor and to test whether the rat vascular endothelium also expresses the CRL receptor protein.

Section snippets

Development and affinity-purification of rabbit polyclonal antibody

A rabbit polyclonal antiserum, MR003, to the carboxy (C)-terminus of rat CRL receptor sequence, SIQDIENVALKPEKLYDLVM (Chang et al., 1993) (Polypeptides Laboratories, Wolfenbuettel, Germany), was raised and affinity-purified according to a previously described scheme (Hagner et al., 2001).

Isolation and culture of rat pulmonary microvascular endothelial cells

Rat lung endothelial cells were prepared from adult Wistar rats by a previously described method (Duijvestijn et al., 1992) that employed a monoclonal antibody specific for rat endothelial cell antigen-1

Results

A cell line that was stably transfected with cDNAs encoding the rat CRL receptor and human RAMP3 enabled us to characterize the rat CRL receptor antibody MR003 by immunofluorescence and Western blot analysis. For the immunohistochemistry analyses, the specificity of immunostaining was established by the absence of immunostaining following pre-incubating the antibody with the peptide antigen (data not shown). Fig. 1A shows that the transfected HEK cells express CRL receptor-immunoreactivity,

Discussion

Here we describe the development and characterisation of an affinity-purified polyclonal antibody for the rat CRL receptor, with which we obtained novel evidence that the rat vascular endothelium expresses the CRL receptor protein as we have shown already in human (Hagner et al., 2002c). Particularly noteworthy is that, similar to our findings in human, the microvascular endothelium appears to express high levels of CRL receptor.

By using immunohistochemistry, we were able to show that our

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