Adrenomedullin (AM) was discovered in the adrenal gland in 1993 by Kitamura and colleagues. Since then it has been found in many other tissues including in skin. We know from our recent studies that AM regulates (in vitro) the secretion of cytokines and growth factors, known to be involved in tissue repair, from endothelial and skin cells. The present study was designed to establish whether AM levels in skin were increased following wounding in vivo.
A well-characterized excisional wounding model was adopted to investigate this. Using a biopsy punch, 4 x 8mm full-thickness incisional wounds were made in the shaven dorsal surface of anaesthetized male Sprague Dawley rats (200-250g). Animals were sacrificed at 1, 2, 3, 4, 6, 12 and 24 hours post-wounding and the area 2 mm adjacent to the wound site was harvested and immediately frozen in liquid nitrogen. Two wounds per animal were prepared for mRNA quantification using kits (Amersham Pharmacia Biotech) and two wounds were extracted for peptide quantification using a SEP-Pak type extraction protocol. Real time PCR was performed (Stratagene MX4000 model) on all samples and AM mRNA values were normalized against the house keeping gene GAPDH. AM peptide levels were measured from skin extracts using a radioimmunoassay kit (Phoenix).
The real-time PCR results demonstrate a time-dependent increase in AM mRNA which becomes significant (P <0.05) at the 3 hour time point. AM peptide levels were significantly elevated at 6hrs post-wounding and had returned to basal by 24 hours. This result demonstrates that AM peptide and gene expression are up regulated in rat skin in a time-dependent manner following wounding and suggests a role for AM in the inflammatory phase of tissue repair.
22 - 24 Mar 2004
British Endocrine Societies