Elsevier

Brain Research

Volume 955, Issues 1–2, 15 November 2002, Pages 64-71
Brain Research

Research report
Paracrine/autocrine function of adrenomedullin in peripheral nerves of rats

https://doi.org/10.1016/S0006-8993(02)03365-6Get rights and content

Abstract

The presence of adrenomedullin (AM) and of an AM receptor were investigated in highly enriched primary cultures of Schwann cells and perineural fibroblasts of newborn and adult rats. AM was released into the conditioned medium of adult perineural fibroblasts (1749±629 pgeq/105 cells per 24 h). mRNA encoding AM was also predominantly expressed in adult perineural fibroblasts. mRNA encoding the calcitonin receptor-like receptor (CRLR) and the receptor-activity-modifying proteins (RAMP) 1, -2 and -3 were demonstrated in all the primary cells, but the levels of RAMP1 mRNA relative to 18s rRNA were 10-fold lower than those of CRLR and RAMP2 and -3 encoding mRNA. The results are consistent with the expression of CRLR/RAMP2 and CRLR/RAMP3 heterodimeric AM receptors in all the primary cells examined. AM stimulated cAMP accumulation in newborn (EC50 0.62±0.29 nM) and adult (EC50 0.45±0.03 nM) rat Schwann cells and in newborn (EC50 0.79±0.50 nM) and adult (EC50 1.06±0.72 nM) rat perineural fibroblasts. The EC50 of calcitonin gene-related peptide stimulated cAMP production was 93- to 100-fold higher than those of AM in the four types of primary cells studied. The co-expression of AM and its receptor in perineural fibroblasts and the expression of an AM receptor in Schwann cells suggest autocrine and/or paracrine modes of action of AM in peripheral nerves.

Introduction

Human and rat adrenomedullin (AM) are 52- and 50-amino acid polypeptides belonging to the calcitonin (CT) family of peptides that also includes CT, α- and β-CT gene-related peptide (CGRP) and amylin [31]. AM, CGRP and amylin share six amino acid ring structures, linked by disulfide bonds between cysteine residues, and amidated C-termini, both required for the biological activity of the peptides. AM and CGRP are potent vasodilatory and hypotensive peptides that interact with receptors on endothelial and vascular smooth muscle cells [22].

Adrenomedullin was first isolated from a pheochromocytoma, but subsequently found in the majority of the tissues examined, consistent with a wide range of biological actions [11], [14].

In the brain, immunoreactive AM is widely distributed. It was recognized in neurons, astrocytes and glial cell tumors [23], [25], [26], [27], [29], and AM encoding mRNA was increased in interferon-γ treated rat astrocytes [15]. AM stimulated the proliferation of several tumor cell lines including those of neuroblastoma or glioblastoma origin [18], [21]. Auto- and/or paracrine modes of action, neutralized with antibodies to AM, have been proposed [21]. Mitogenic activity of AM was also observed in AM producing retinal pigment epithelial cells [28]. AM-provoked proliferation of Swiss 3T3 fibroblasts is mediated by cAMP/PKA dependent signal transduction [32]. In rat renal mesangial cells, on the other hand, AM inhibited [3H]thymidine incorporation and suppressed proliferation of quiescent and PDGF stimulated cells [6], [24].

Adrenomedullin receptors linked to cAMP formation have been revealed in mouse and rat fibroblast cell lines, in rat astrocytes in primary culture and in the human oligodendroglial cell line KG1C [7], [13], [30], [33].

An initially orphan CT receptor-like receptor (CRLR) of the B family of G protein-coupled receptors with seven transmembrane domains requires co-expression of associated receptor-activity-modifying proteins (RAMP) for functional expression [17]. A CRLR/RAMP1 complex is a CGRP receptor. However, with RAMP2 or -3 the CRLR forms AM receptors [3], [8], [12]. Along these lines, RT-PCR analysis revealed expression of mRNA encoding the CRLR and RAMP1, -2 and -3 in the human oligodendroglial KG1C cell line [30]. Both the CLRL/RAMP1 and -2 defined CGRP and AM receptors are coupled to cAMP production [3], [17].

Perineural fibroblasts and Schwann cells are non-neuronal resident cells in peripheral nerves required for the formation of the extracellular matrix, and the basal lamina and myelin sheath, respectively (for review see Ref. [9]). Both Schwann cells and fibroblasts proliferate after nerve injury, and Schwann cells remain the predominant cell type in vacated endoneural tubes. Local interactions among neurons, Schwann cells and perineural fibroblasts are critical for proper development and regeneration of the peripheral nervous system.

Here, we have demonstrated the release of AM from perineural fibroblasts, and have identified AM receptors in Schwann cells and perineural fibroblasts. Taken together the results imply local signaling of AM in perineural fibroblasts and Schwann cells of peripheral nerves.

Section snippets

Materials

Rat (r) AM and rCGRP were supplied by Phoenix Pharmaceuticals (Belmont, CA, USA). Recombinant heregulin-β1 was donated by M. Sliwkowski (Genentech, South San Francisco, CA, USA). Plastic ware for tissue culture was purchased from Becton Dickinson (Basel, Switzerland) and culture media, fetal calf serum (FCS) and B27 supplemented mixture from Life Technologies (Basel, Switzerland). Trypsin was purchased from Worthington Biochemicals (Lakewood, NJ, USA) and collagenase and dispase from Roche

Cell characterization

Potential cross-contamination of fibroblasts and Schwann cells kept in primary culture was analyzed by immunocytochemistry and FACS. Adherent Schwann cells displayed characteristic bipolar spindle-like shapes and stained for the Schwann cell specific immunoreactive low affinity NGF-receptor (Fig. 1). Perineural fibroblasts were flat with a polygonal shape and stained for the Thy 1.1 marker protein. Both newborn and adult rat perineural fibroblasts were enriched after three passages to between

Discussion

An understanding of mechanisms involved in the development and regeneration of peripheral nerves is a prerequisite for successful reconstruction of their lesions. Here, AM was released from adult perineural fibroblasts. The expression of AM receptors in the same cells and in newborn and adult Schwann cells suggests local regulatory roles of AM in peripheral nerves. Endothelial and smooth muscle cells of blood capillaries surrounding peripheral nerves are potential additional sources of AM

Acknowledgements

The authors thank Dr M. Sliwkowski (Genentech, South San Francisco, CA, USA) for the heregulin. This work was supported by the Swiss National Science Foundation, the University of Zurich and the Schweizerische Verein Balgrist.

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