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alpha-Neo-endorphin and dynorphin immunoreactivities in rat brain were visualized by double antibody immunofluorescence of frozen sections. Antibodies were used that were specific for their respective antigens. The pattern of neuronal immunostaining produced by alpha-neo-endorphin and dynorphin antisera in adjacent serial sections was completely superimposible. No areas were found in which alpha-neo-endorphin or dynorphin immunoreactivities existed alone. The following brain regions contained alpha-neo-endorphin/dynorphin-immunoreactive fibers and terminals: the median forebrain bundle, the internal capsule, the substantia nigra, the hypothalamus, the nucleus accumbens, the hippocampus, and the medulla oblongata. A few fibers were seen in the cerebral cortex and in the corpus striatum. In many regions, this neuronal fiber system seems to overlap the neuronal system previously described to contain [Met]-/[Leu]enkephalin-immunoreactive material. In brains from colchicine-treated animals, numerous alpha-neo-endorphin/dynorphin-immunoreactive neuronal cell bodies were seen in the supraoptic, retrochiasmatic supraoptic, paraventricular, and magnocellular accessory nuclei of the hypothalamus. It is concluded that alpha-neo-endorphin-like and dynorphin-like immunoreactivities are part of the same neuronal system.

Peptides derived from both proenkephalin and prodynorphin have been identified in guinea pig adrenal medulla. In extracts of whole adrenal glands radioimmunoassays directed to the prodynorphin-derived peptides alpha-neoendorphin, dynorphin A, and dynorphin B detected high concentrations of immunoreactive material ranging from 113 to 216 pmol/gm. The concentrations measured by radioimmunoassays directed to the proenkephalin products met-enkephalin-Arg-Gly-Leu and met-enkephalin-Arg-Phe were 878 and 484 pmol/gm, respectively. No metorphamide or dynorphin(1-8) could be detected in the adrenals. Leucine-enkephalin immunoreactivity which can be generated from either prodynorphin or proenkephalin could also be measured in the extracts. Gel filtration showed the immunoreactive material, with the exception of that measured by the alpha-neoendorphin radioimmunoassay, to be predominantly of high molecular weight ranging from Mr = 3,000 to 12,000. Immunocytochemistry, using well characterized antisera to alpha-neoendorphin and met-enkephalin-Arg-Gly-Leu, demonstrated that the prodynorphin and proenkephalin products were present in the same cells in the medulla region of the gland. The results show that two opioid peptide precursors can be localized in the same cells and exhibit some common features in their processing. As a relatively homogeneous, localized system, the guinea pig adrenal gland should prove a valuable, in vivo model for the study of co-localized opioid precursors.

BAM 18 is a derivative of the opioid precursor proenkephalin A. Although it exists in rat and guinea-pig brain in relatively high concentrations, its physiological function is presently unknown. In the present study we have determined the opioid receptor selectivity of this peptide using radioligand binding and peripheral tissue bioassay. When selective binding conditions were used, BAM 18 bound to the mu opioid receptor with an affinity three times that of the kappa opioid receptor and over 10 times that of the delta opioid receptors (Ki = 0.29, 0.75, and 3.2 nM respectively). BAM 18 also displayed mixed receptor selectivity in in vitro bioassay. Ke values for naloxone antagonism of BAM 18 agonist activity in the electrically stimulated guinea-pig ileum and the mouse vas deferens were 4.3 and 9.9 nM, respectively. These data indicate that BAM 18 binds to all three opioid receptor subtypes with a selectivity profile of mu greater than kappa greater than delta.

Antibodies have been raised to a synthetic peptide corresponding to the C-terminal 15-amino acid residues of prodynorphin, the common precursor to the neo-endorphins and dynorphins. The amino acid sequence of the antigen was based on the sequence deduced from mRNA isolated and cloned from porcine hypothalamus (Kakidani, H., Y. Furutani, H. Takahashi, M. Noda, Y. Morimoto, T. Hirose, M. Asai, S. Inayama, S. Nakanishi, and S. Numa (1982) Nature 298: 245-248). Using a radioimmunoassay developed from these antibodies we have isolated an endogenous prodynorphin C-fragment from bovine caudate nucleus. The isolated peptide displayed characteristics on gel filtration similar to those of synthetic prodynorphin C-fragment predicted from the porcine mRNA sequence but had low cross-reactivity in the radioimmunoassay. Sequencing and amino acid analysis showed a substitution of serine for asparagine at position 6 in the porcine sequence. Dynorphin B (rimorphin), which is adjacent to prodynorphin C-fragment in the precursor, was isolated from the same extract. Amino acid analysis and elution position on a gel filtration column confirmed its structure as that previously characterized from bovine pituitary extracts. The release of prodynorphin C-fragment and the C-terminus of dynorphin B from the porcine precursor would require cleavage at a single arginine residue. However, a terminal arginine was not present on either of these prodynorphin peptides isolated from bovine caudate. The data would suggest that processing at a single arginine residue results in elimination of the arginine, a feature in common with processing at paired basic residues.

Extracts from adult human adrenals contained high concentrations of immunoreactive beta-endorphin and alpha-melanotropin. Lower quantities of immunoreactive adrenocorticotropic hormone could also be detected. Distribution studies showed the presence of pro-opiomelanocortin fragments in the adrenal medulla. No alpha-melanotropin, beta-endorphin, or adrenocorticotropic hormone could be found in adrenal extracts from several other mammalian species. Analysis of the beta-endorphin-like immunoreactivity using region specific radioimmunoassays interfacing with gel filtration and reverse-phase high-performance liquid chromatography showed the majority of the beta-endorphin-like material to exist as nonacetylated beta-endorphin-(1-31) with a small percentage of lipotropin-sized molecules. The alpha-melanotropin-like immunoreactivity cochromatographed on gel filtration and reverse-phase high-performance liquid chromatography with desacetyl alpha-melanotropin. The data suggest that pro-opiomelanocortin is expressed in the adrenal medulla of humans but is not detectable in the adrenal glands of many other mammalian species.

Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys-Arg) typical processing site, would generate two opioid fragments, the amino-terminal fragment BAM 18 and the carboxy-terminal fragment Leu-enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu-enkephalin). Here we present evidence that BAM 18 and Leu-enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys-Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu-enkephalin was found to be present in much greater amounts than was BAM 18-like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu-enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu-enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu-enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.

Dehydration significantly reduced the concentration of immunoreactive dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, beta-neo-endorphin, and leu-enkephalin in the rat pituitary posterior-intermediate lobe. A statistically significant increase in immunoreactive dynorphin A(1-8), alpha-neo-endorphin and leu-enkephalin was observed in the hypothalamus. Comparison of the molar ratios of dynorphin A(1-17): dynorphin A(1-8) and alpha-neo-endorphin: beta-neo-endorphin showed an altered profile of stored pro-dynorphin cleavage products in the posterior-intermediate lobe of the pituitary of dehydrated rats.

BAM-18, a new endogenous opioid containing 18 amino acid residues, was tested in 3 behavioral paradigms. Tail-flick analgesia, a spinally mediated response, hot-plate analgesia, a centrally mediated response, and open-field locomotor activity. Rats were stereotaxically implanted with a unilateral cannula aimed at the lateral ventricle. Following recovery, each animal was tested in one of the paradigms after receiving an intraventricular injection of BAM-18, morphine or the Ringer's vehicle. BAM-18 produced significant tail-flick analgesia only at doses (50 micrograms) 50 times higher than those needed with morphine (1 microgram). BAM-18 produced an extended hyperalgesia at lower doses (5 micrograms) that was also seen transiently at the high dose. The analgesia but not the hyperalgesia was reversed by naloxone (10 mg/kg, s.c.). BAM-18 produced significant naloxone-reversible hot-plate analgesia, but again it was less potent than morphine (50 micrograms for BAM-18 vs. 5 micrograms for morphine). There was no evidence of hyperalgesia in this paradigm. Locomotor activity, following 50 micrograms of BAM-18, resembled control injections for the first 18 minutes, then became reduced in a manner similar to morphine (5 micrograms). This reduction in activity was completely reversed by naloxone. These data suggest that BAM-18 is indeed an opioid molecule but is at least 10 times less potent at altering behavior than morphine.

The opioid receptor selectivity of BAM 18 was determined by radioligand binding and peripheral tissue bioassay. Using selective radioligand binding conditions, BAM 18 bound to the mu opioid receptor with an affinity twice that of the K receptor and over 10 times that of the delta opioid receptor (Ki = 0.29, 0.84 and 3.9 nM, respectively). Ke values for naloxone antagonism of BAM 18 activity in the electrically stimulated guinea pig ileum and the mouse vas deferens were 4.3 and 9.9 nM respectively. The pharmacological profile of BAM 18 was similar to that of metorphamide.