Dynorphin immunocytochemical localization in brain and peripheral nervous system: preliminary studies.

Abstract

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 ...

Specific processing of the thyrotropin-releasing prohormone in rat brain and spinal cord

“…Alternatively the resistance to cleavage exhibited by the basic residues that follow the TRH tripeptide may be due to the presence, in three out of five cases, of paired arginine residues in contrast to the lysylarginine residues which precede the TRH sequences. It may be relevant to observe that the arginylarginine sequence at positions 6 and 7 in the opioid peptide dynorphin remains intact during the elaboration of the peptide whereas the lysylarginine residues on each side of the dynorphin sequence undergo cleavage [13]. This would suggest that the enzymes that are involved in cleavage at LysArg sites are different from the enzymes that have specificity for Arg-Arg sequences.…”

The effect of naloxone on vasopressin release from rat neurohypophysis incubated in vitro.

“…Thus, the endogenous opiate which reduces vasopressin release from the neurointermediate lobe appears to originate in the intermediate lobe of the pituitary gland. This part of the hypophysis is virtually devoid of dynorphin (Watson et al 1981), contains only small amounts, if any, of enkephalin (Rossier et al 1979) but high concentrations of peptides exhibiting /?-endorphin-like immunoreactivity (Pelletier et al 1977;Bloom et al 1977). The presence of biologically active fl-endorphin-(1-31) has been revealed by chromatographic techniques, notwithstanding that the forms of fl-endorphin which predominate are those which have undergone proteolysis or acetylation (Smyth & Zakarian, 1980;Seizinger & H6llt, 1980;Weber, Evans, Chang & Barchas, 1982).…”

“…The posterior pituitary gland possesses a high density of opiate binding sites (Simantov & Snyder, 1977). Opioid peptides have been found in the blood (Guillemin, Vargo, Rossier, Minick, Ling, Rivier, Vale & Bloom, 1977; Clement-Jones, Lowry, Rees & Besser, 1980) and in the neurohypophysis (Rossier, Battenberg, Pittman, Bayon, Koda, Miller, Guillemin & Bloom, 1979;Watson, Akil, Ghazarossian & Goldstein, 1981). They might be the endogenous ligands of the opiate receptors in the pars nervosa.…”

Opioid receptor types on adrenergic nerve terminals of rabbit ear artery

“…The physiological significance of enkephalins and other opioid peptides as modulators of neuronal activity has recently been suggested from the findings that these peptides are located in or near nervous structures Schultzberg et al, 1979;Watson et al, 1981) and are released in response to nerve stimulation (Schulz et al, 1977;Corbett et al, 1980). Moreover, there is a report that enkephalins depress the output ofacetylcholine (ACh) and noradrenaline, possibly by a presynaptic action in various nervous systems .…”