Characterization of enkephalins in rat adrenal medullary explants

Abstract

In the rat, removal of depolarizing stimuli to the adrenal medulla by surgical denervation in vivo or by explanting adrenal medullae has been shown to dramatically increase preproenkephalin mRNA, and enkephalin-containing (EC) peptides. To further elucidate the cellular basis of these effects and the role of transsynaptic influences on post-translational processing, we have defined the time course, and characterized EC peptides in rat adrenal medullary explants in control and depolarized states. The rise in EC peptides begins after 1 day in culture and reaches a peak at 4–7 days. Although the onset of the increase in EC peptides in culture is delayed by 12–24 h compared to the changes seen in vivo, following surgical denervation, the time course of peak and duration is remarkably similar. Size exclusion chromatography (SEC) revealed that the major species of newly appearing EC peptides in explanted glands is a high molecular weight peptide of approximately 18,000 with a Met-/Leu-enkephalin ratio of approximately 6. These results suggest that proenkephalin, the initial precursor of the EC peptide family, is the major EC peptide that accumulates in rat adrenal medullary explants. A low-molecular weight EC peptide, found by high-performance liquid chromatography to be free Met-enkephalin, is a minor component of the culture induced increase in EC peptides. Culturing of medullae in the presence of depolarizing concentrations of K + prevents the accumulation of the proenkephalin-like EC peptides and free enkephalins. Thus, both denervated and explanted rat adrenal medullae accumulate proenkephalin which, in turn, is subject to regulation by depolarizing stimuli. However, limited processing to smaller enkephalin peptides occurs in culture, and is largely unaffected by depolarizing stimuli.