Up-regulation of adrenomedullin gene expression in the regenerating rat adrenal cortex

Adrenomedullin (AM) is an endogenous regulatory peptide that exerts growth-promoting action in several normal and neoplastic tissues, and we investigated whether its gene expression changes during rat adrenal regeneration after enucleation and contra-lateral adrenalectomy. Regenerating adrenals were collected at day 0 (just after enucleation; control rats), 1, 3, 7, 14 and 28 after surgery. The immunocytochemical assay of PCNA (proliferating cell nuclear antigen) index confirmed that the early stages of regeneration can be divided into an initial differentiation period (from day 0 to day 3) and a subsequent high proliferative period (days 5 and 7) followed by a decrease in the proliferation activity. Real time-polymerase chain reaction (PCR) demonstrated that AM mRNA expression underwent a marked rise at day 1 of regeneration, attained a maximum level at days 3 and 5, and then declined from day 7, returning to the control value at days 14 and 28. Western blotting showed that AM protein expression was moderately elevated at day 1, was maximal between days 3 and 7, and then decreased at days 14 and 28, although remaining significant over the control value. Taken together, our findings indicate that the increase in the AM gene transcription and translation may be considered one of the early events in the enucleation-induced activation of local adrenocortical stem cells, conceivably favoring both the differentiation and proliferation stages of regeneration. The mechanism underlying this adrenocortical stem cell response does not seem to involve ACTH, because real time-PCR demonstrated that it also occurred in animals whose contralateral adrenal glands were spared, and consequently the level of circulating ACTH was in the normal range. It remains to be investigated whether the enucleation-induced relative hypoxia, ensuing from disruption of the vascular bed, and the local release of inflammatory cytokines may be involved in the up-regulation of AM gene expression in regenerating adrenal glands.