In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.
Immobilization (IMMO) stress was used to examine how stress alters the stress hormone epinephrine (EPI) in the adrenal medulla in vivo. In rats subjected to IMMO for 30 or 120 min, adrenal corticosterone increased to the same extent. In contrast, EPI changed very little, suggesting that EPI synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological responses of the 'flight or fight' response. In part, stress activates EPI via the phenylethanolamine N-methyltransferase (PNMT) gene as single or repeated IMMO elevated PNMT mRNA. The rise in PNMT mRNA was preceded by induction of the PNMT gene activator, Egr-1, with increases in Egr-1 mRNA, protein, and protein-DNA binding complex apparent. IMMO also evoked changes in Sp1 mRNA, protein, and Sp1-DNA complex formation, although for chronic IMMO changes were not entirely coincident. In contrast, glucocorticoid receptor and AP-2 mRNA, protein, and protein-DNA complex were unaltered. Finally, IMMO stress elevated PNMT protein. However, with seven daily IMMOs for 120 min and delayed killing, protein stimulation did not attain the highly elevated levels expected based on mRNA changes. The latter may perhaps suggest initiation of adrenergic desensitization to prolonged and repeated IMMO stress and/or dissociation of transcriptional and post-transcriptional regulatory mechanisms.
AP‐2 is a vertebrate transcription factor expressed in neural crest cells and their derivative tissues, including the adrenal medulla, where epinephrine is produced. AP‐2 is shown to stimulate expression of the gene encoding the epinephrine biosynthetic enzyme phenylethanolamine N‐methyltransferase (PNMT). However, stimulation of the PNMT gene by AP‐2 requires glucocorticoids and appears to be mediated through the interaction of AP‐2 with activated type II glucocorticoid receptors. Mutation of AP‐2 and/or glucocorticoid receptor binding elements within the PNMT promoter disrupts the ability of AP‐2 and glucocorticoids to induce PNMT promoter activity. These findings suggest, in the case of PNMT, that AP‐2 stimulates gene expression through a novel glucocorticoid‐dependent mechanism.
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