Immunoreactive dynorphin (ir-Dyn), immunoreactive leucine-enkephalin (ir-Leu-Enk) and various other neuropeptides were measured in acid extracts of bovine adrenal medulla and isolated adrenal chromaffin cells. Their respective levels ranged as follows: Leu-Enk > Dyn > bombesin > vasoactive intestinal peptide (VIP) > neurotensin > substance P. Comparisons of the total catecholamine levels with the levels of Leu-Enk in both extracts gave ratios in the same order of magnitude (2600, tissue extract and 5000, cell extract). However, the catecholamine/Dyn ratio in the tissue extract (138 000) was much higher than that found in the cell extract (20 180), suggesting a possible selective degradation of Dyn in tissue extract as compared with cell extract or an induction of Dyn biosynthesis in cells which have been isolated from their natural microenvironment. Immunofluorescence staining of isolated chromaffin cell sections revealed the presence of ir-Dyn in 5 to 10% of the total cell population. To localize ir-Dyn in regard to Leu-Enk and catecholamines, adrenal chromaffin cells were separated into three populations (I, II, and III) on a stepwise bovine serum albumin (BSA) gradient. Relative high levels of ir-Dyn were measured in cell layer I (4 pmol/106 cells), a cell population enriched in noradrenaline. However, ir-Leu-Enk was more concentrated in cell layers II and III (5.3 and 8.3 pmol/106 cells), two populations enriched in adrenaline. Isolation and high pressure liquid chromatography (HPLC) analysis of adrenomedullary Dyn indicated the presence of at least five molecular forms corresponding to Dyn-(1-11), Dyn-(1-12), Dyn-(1-13), Ala-containing-Dyn-(1-13) and a nonidentified molecule eluting closely to Dyn-(1-13). These data indicate that adrenal ir-Dyn and ir-Leu-Enk have distinct cellular distributions. In addition, the identification of Dyn fragments in bovine adrenal medulla indicates that these short peptides may be considered as natural active forms of Dyn.
