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ACTH Production in C57BL/6 Mouse Skin

The presence of ACTH peptide in anagen-VI skin was demonstrated by reversedphase (RP) HPLC analysis 7 combined with specific radioimmunoassay (RIA). The major immunoreactive (IR) peak had the same retention time (18–19 min) as the synthetic ACTH standard. No specific IR was detected at the retention time for the desacetylα -MSH (13 min), α -MSH (15 min), diacetylα -MSH (17 min), or β -endorphin (21 min). The ACTH concentration, that was low in telogen (3.06 + 3.59 pg/mg protein), increased during anagen in two steps: a rapid phase in anagen-I, and a slower rise that reached its peak in anagen-VI (107.6 + 8.4 pg/mg protein). By indirect immunofluorescence, ACTH-IR was detected in keratinocytes of basal layer of epidermis, of proximal and distal ORS of hair follicle, and in subcutaneous muscle of anagen-VI skin. Northern hybridization techniques showed the production of 4.5and 2.0-Kb mRNAs hybridizable to the coding region of the MC2 (receptor for ACTH) gene (see F IGURE 1). The expression of both transcripts was similar through the entire hair cycle. 8 To study the effect of ACTH we applied a previously established skin organ culture model. 9 At physiological doses (10 –9 M) ACTH stimulates DNA synthesis in dermal ( p < 0.01) but not in epidermal ( p > 0.05) compartments, and at pharmacological doses (10 –6 –10 –7 M) it inhibited DNA synthesis in both dermis and epidermis ( p < 0.01 and p < 0.001, respectively). Previous organ culture studies have documented that 3 H-thymidine (Tdr) incorporation into epidermal and dermal compartments represented almost exclusively DNA synthesis by epidermal and follicular