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Dynorphin is a recently discovered peptide belonging to the family of endogenous opioids (1). The first five amino acids at the NH2 terminus of dynorphin are identical to leucine-enkephalin; however, from position 6 to 13 it has a unique structure (the remainder ofthe peptide is currently unknown). While the anatomical localization of 3-endorphin and the enkephalins have been well described (2-19), little is currently known about the distribution of dynorphin. This peptide was extracted from porcine pituitaries and was purified by means ofa bioassay using the guinea pig ileum. We therefore elected to begin our studies on dynorphin immunocytochemistry by looking at rat pituitary and brain and at guinea pig ileum. We also extended our studies to guinea pig adrenal because of the previous reports of an enkephalin-like immunoreactivity in that organ. MATERIALS AND METHODSTissue preparation for immunocytochemistry was as described (17). Antisera against dynorphin-(1-13) were prepared, with the resulting antigenic specificity largely directed toward a COOHterminal region (20). Several antisera were produced, with similar specificities; that of highest titer (Lucia) was chosen, because it has been the most thoroughly characterized for radioimmunoassay (RIA). As published elsewhere (20), the Lucia antiserum is used in RIA at titers of from 1:50,000 to 1:200,000and crossreacts with leucine-enkephalin less than 10-8, and [3H]leucine-enkephalin does not appear to bind even in dilutions as low as 1:30. For peroxidase-antiperoxidase immunocytochemistry this antiserum was used at a 1:600 dilution for a 24-hr incubation at 40C (peroxidase-antiperoxidase and related reagents were from Sternberger-Meyer Immunocytochemical, Jarrettsville, MD)..An important issue in the immunocytochemical study of dynorphin distribution in brain is the potential confusion with leucine-enkephalin-containing structures. The antiserum used in this study (Lucia) was raised against dynorphin-(1-13) (Peninsula Labs, San Carlos, CA) and its antigenic determinant includes residues 4 and 5 of dynorphin and leucine-enkephalin. While there is little crossreactivity with leucine-enkephalin under RIA conditions, the Lucia antiserum was used in a much more concentrated form for immunocytochemistry. It is therefore possible that the lower-affinity populations ofantibody may come into play under our immunocytochemical conditions, and that the crossreactivity with other peptides, including leucineenkephalin, may be different from that observed in RIA. For that reason complete control blocking studies were carried out on all tissues, using 2-20 A.M concentrations ofmethionine-and leucine-enkephalin, 8-endorphin, oxytocin, vasopressin, and dynorphin-(1-13).In experiments in which relative concentrations ofdynorphin and enkephalin were determined, a combined high-performance liquid chromatography (HPLC)-RIA procedure was employed. All tissue was obtained fresh after decapitation, kept chilled on ice during dissection, then frozen immediately on dry ice and ...

Distribution of the potent opioid peptide dynorphin has been determined in pituitary gland (pig, beef, rat), in the various regions of rat brain, and in rat spinal cord, by using a highly specific antiserum. By gel permeation chromatography in 4 M guanidine, the porcine pituitary immunoreactivity is found in a major peak of apparent molecular weight about 1700 and a minor peal of about 3400. Similar peaks are found in rat pituitary extracts, whereas rat brain contains, in addition, two peaks of larger apparent molecular weight. In the pituitary, immunoreactive dynorphin is found predominantly in pars nervosa. In the central nervous system, it is distributed widely, with highest concentrations in hypothalamus, medulla-pons, midbrain, and spinal cord. Although dynorphin contains leucine-enkephalin, the regional distribution of dynorphin is different from that of enkephalin or of any other known opioid peptide.Dynorphin is an opioid peptide, recently isolated from porcine pituitary extract, which contains leucine-enkephalin at its NH2 terminus. It is distinguished by its extraordinary potency in the guinea pig myenteric plexus-longitudinal muscle bioassay (1). Its first 13 residues are Tyr-Gly-Gly-Phe-Leu-Arg-Arg-IleArg-Pro-Lys-Leu-Lys. Dynorphin-(1-13) was as potent as natural dynorphin in the bioassay. Antisera were raised against thyroglobulin-conjugated dynorphin-(1-13), and one of them ("Lucia 9/14") was used as the basis for a sensitive and highly specific radioimmunoassay (RIA) (2). For significant immunoreactivity, this antiserum requires residues in addition to those of the leucine-enkephalin portion of the peptide; crossreactivity of leucine-enkephalin itself is less than 10-6%. Moreover, no other opioid peptide crossreacts significantly. The free carboxyl group of Lys-13 is not essential for immunoreactivity, because both dynorphin-(1-12) and the methyl ester of dynorphin-(1-13) are fully immunoreactive. Therefore, naturally occurring dynorphin, which is thought to have at least four additional residues at the COOH terminus (3), should be measurable. Dynorphin-(2-13) is also highly immunoreactive, indicating that a free amino group at Tyr-1 is not essential for immunoreactivity, and therefore that precursor peptides with NH2-terminal extensions should also be measurable. We now report the distribution of immunoreactive (ir-) after careful removal of excess fluid and extracted. The dissection was as described by Glowinski and Iversen (4), but with the following modifications. Hypothalamus was dissected out as a uniform rectangular block approximately 4.0 mm wide, 5.5 mm long, and 2.5 mm thick, weighing about 55 mg. The lateral bounds were straight cuts in the sulcus between hypothalamus and adjacent cortex, and the dorsal cut was just ventral to anterior commissure. Although the resulting block of tissue was smaller than that obtained by Glowinski and Iversen, it had the dimensions of hypothalamus as described by Craigie (5). Hypothalamus was divided into anterior and posterior halves by a perpe...