Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat 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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2004, Biochemical and Biophysical Research CommunicationsCitation Excerpt :The membrane was saturated for 2 h in TBS containing 0.1% Tween and 5% non-fat dried milk. A rabbit polyclonal antiserum, MR003, to the carboxy-terminus of rat CRLR was raised and affinity-purified according to a previously scheme [7,8]. The primary rabbit polyclonal antibodies against RAMP1 (sc-11379, Santa Cruz Biotechnology, Heidelberg, Germany), RAMP2 (sc-11380, Santa Cruz Biotechnology, Heidelberg, Germany), and RAMP3 (sc-11381, Santa Cruz Biotechnology, Heidelberg, Germany) were used at 1:500 dilution.
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