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Neoendorphin-Derived Peptides in Rat Hippocampus (Opioid Peptide/In Vitro Neurotransmitter Release/HPLC Resolution) C

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Neoendorphin-Derived Peptides in Rat Hippocampus (Opioid Peptide/In Vitro Neurotransmitter Release/HPLC Resolution) C Proc. Nati. Acad. Sci. USA Vol. 80, pp. 7669-7673, December 1983 Neurobiology Relative contents and concomitant release of prodynorphin/ neoendorphin-derived peptides in rat hippocampus (opioid peptide/in vitro neurotransmitter release/HPLC resolution) C. CHAVKIN*, C. BAKHIT*, E. WEBERt, AND F. E. BLOOM* *A. V. Davis Center, Salk Institute, La Jolla, CA 92037; and tDepartment of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA 94305 Contributed by Floyd E. Bloom, August 24, 1983 ABSTRACT The contents and molecular forms of five dif- hippocampal pyramidal cells (17, 18). The anatomical organi- ferent prodynorphin-derived opioid peptides were compared in zation of the hippocampus makes it accessible to electrophysio- extracts of rat hippocampus by radioimmunoassay after Ca8-HPLC logic recording and permits analysis of the function of the en- resolution., Dynorphin (Dyn) A(1-17) immunoreactivity (ir) and Dyn dogenous prodynorphin-derived opioids. In vivo (19, 20) and in B-ir were heterogeneous in form; Dyn A(I-8)-ir, a-neoendorphin vitro (21) studies have shown complex effects of Dyn A(1-17) (aneo)-ir and 3-neoendorphin (Pneo)-ir each eluted as single ho- on the firing rates and-excitability of hippocampal pyramidal mogeneous peaks of immunoreactivity. The fraction of immu- cells. The high density of dynorphin/neoendorphin-ir fibers in noreactivity having the same retention as the appropriate syn- this region and the electrophysiologic effects of administered thetic standard was used to estimate the actual hippocampal content Dyn A(1-17) suggest that the prodynorphin-derived opioid of each peptide. Comparison of these values showed that the con- centrations of Dyn B, aneo, and Dyn A(1-8) were nearly equal, peptides may function in the control of neuronal excitability whereas both Dyn A(1-17) and .neo were 1/5th to 1/10th the value within the hippocampus. of the other three. Calcium-dependent KV-stimulated release of An understanding of a possible neurotransmitter role for the these prodynorphin-derived opioids from hippocampal slices was prodynorphin-derived opioids in this brain region requires the detected. The stimulated rates of release were highest for Dyn B- demonstration that these peptides are present within the neu- ir followed by aneo-ir, then (3neo-ir and Dyn A(1-8)-ir with Dyn rons in bioactive form and can be released at nerve terminals A(I-17)-ir lowest. The relative rates of stimulated release were in after appropriate stimulation. In the present study, we sought agreement with the relative proportions of peptide present within to determine the relative abundance of the prodynorphin-de- the tissue. This evidence of the presence and release of these opioid rived peptides within the hippocampus and to relate these val- peptides considerably strengthens the hypothesis that this family ues of stored peptide to the relative distribution of peptide forms of endogenous opioids plays a neurotransmitter role in the hip- that can be released in vitro from hippocampal slices. pocampus. METHODS The polypeptide sequence of the dynorphin/neoendorphin precursor (prodynorphin) was recently deduced by Kakidani et To prepare- tissue extracts, hippocampi from male Sprague- al. (1) using cDNA cloning techniques. Three [Leu5]enkephalin Dawley rats (180-220 g) were rapidly dissected, trimmed of segments, each having a unique COOH-terminal extension, are subiculum, suspended in 10 vol of hot (90'C) 1 M acetic acid, contained in this sequence (Fig. 1). Based on peptide purifi- heated in a boiling water bath for 30 min, sonicated 15 see (Kontes cation and radioimmunoassay (RIA) results, it is thought that probe sonicator), and then centrifuged for 60 min at 20,000 x the post-translational processing of prodynorphin may yield at g at 4.C. Acid-insoluble protein was assayed by the Lowry pro- least five different opioid peptide products. -a-Neoendorphin cedure (22). Acid-soluble extracts were lyophilized and sus- (aneo) and S-neoendorphin (,Bneo), originally described by pended in a minimal volume of methanol/0. I M HC1, 1:1 (vol/ Matsuo and co-workers (2, 3), overlap within the first COOH vol). terminally extended [Leu5]enkephalin segment. Dynorphin Radioimmunoassays were carried out on tissue extracts and (Dyn) A(1-17), isolated by Goldstein et al. (4, 5) and its frag- column fractions using specific antisera as described (8, 23-25). ment Dyn A(1-8) (6-8) are present within the second segment. Peptides were iodinated as described (23) and then purified by The third opioid segment in prodynorphin (Dyn B) corresponds C18-HPLC. The RIA protocol was as follows: 200 Alp of tracer to the 13-amino acid peptide sequence independently deter- (5,000 cpm) diluted in buffer A (150 mM sodium phosphate, pH mined by Fischli et al. (9) and by Kilpatrick et al. (10). This pro- 7.4/0.1% bovine serum albumin/0.1% Triton X-100) and 100 dynorphin-derived peptide family together with the proen- .ul of antiserum diluted in buffer A were added to a 100-.1u sam- kephalin products and pro-opiomelanocortin opioid products ple diluted in 1.0% Triton X-100/0. 1 M HC1. This mixture was compose the three distinct groups of endogenous opioids (11, incubated for 18-24 hr at 40C; antibody-bound peptide was 12). separated by the dextran-charcoal procedure (23). Each of the The identified prodynorphin products [Dyn A(1-17), Dyn antisera used was highly selective; crossreactions with other en- A(1-8), Dyn B, aneo, and fneo] are widely distributed in brain, dogenous opioid peptides were not appreciable (8, 23-25). Opioid spinal cord, and peripheral autonomic ganglia and form a dis- peptides were synthesized and purified by N. Ling (Salk In- tinct opioid system (13-16). Within the rat hippocampus Dyn stitute). A immunoreactivity (ir) is densely concentrated in the granule For release experiments, hippocampal tissue was rapidly dis- cell nerve fiber projection (the mossy fibers) innervating the sected and then sliced in two perpendicular planes at 250-,tm thickness using a McIlwain tissue chopper. The slices were The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: RIA, radioimmunoassay; ir, immunoreactivity; Dyn, dy- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. norphin; aneo, a-neoendorphin; Vneo, (3-neoendorphin. 7669 Downloaded by guest on September 30, 2021 7670 Neurobiology: Chavkin et al. Proc. Natl. Acad. Sci. USA 80 (1983) a-NEO-ENDORPH IN 171 B-NEO-ENDORPHIN FIG. 1. COOH-terminalportionofporcinepro- .... GLN VAL LYS ARG TYR GLY GLY PHE LEU ARG LYS TYR PR6 LYS ARG SER SER - dynorphin sequence (residues 171-256) deduced by Kakidani et-al. (1). The three (Leu'lenkephalin- containingsegments are bracketedby the putative GLU VAL ALA GLY GLU GLY ASP GLY ASP ARG ASP LYS VAL GLY HIS GLU ASP LEU TYR - peptide processing signal 'Lys-Arg" (1). aneo and I IDYNORPHIN A(1-17) (3neo overlap asprodynorphin residues 175-184 and LYSARGF- DYNORPHIN A(1-8) ------ |6 175-183, respectively. Dyn A(1-17) corresponds to LYS ARG TYR GLY GLY PHE LEU ARG ARG IL ARG PRO LYS LEU LYS TRP ASP ASN G the full sequence ofthe second segment. Dyn A(1- 8) is produced by an unusual cleavage to the left of f DYNORPHIN B(1-13) arginine-217. A similar cleavage to the left of ar- LYS ARG TYR GLY GLY PHE LEU ARG ARG GLN PHE LYSTHIVAL VAL ARG SER GLN GLU - ginine-241 produces Dyn B from the third [Leu5]- enkephalin-containing segment. The presence of the full sequence ofDyn B(1-29) in brain tissue has ASP PRO ASN ALA TYR TYR GLU GLU LEU PHE ASP VAL - OH not yet been reported. washed three times with a Krebs-bicarbonate solution modified molecular weight of 3,500-4,000, which is consistent with its to contain the following: 127 mM NaCl/3.85 mM KCI/1.8 mM longer retention on C18-HPLC and a greater hydrophobicity. CaCl2/1.8 mM KH2PO4/1.18 mM MgSO4/20 mM NaHCO3/ The high molecular weight and equal crossreactivities in Dyn 11 mM D-glucose/bovine serum albumin (1 mg/ml) (Miles, A(1-17) and Dyn B RIAs indicate that this material may be Dyn crystalline)/bacitracin (Sigma) at 30 ug/ml and bubbled with A(1-32), an intermediate precursor of both Dyn A(1-17) and 5% C02/95% 02 (vol/vol) for at least 30 min (pH 7.4). Slices Dyn B described by Fischli et al. (9). For each peptide, the frac- were placed in a 1-inch diameter (1 inch = 2.54 X 10-2 m) su- tion of the total immunoreactivity eluting from C18-HPLC hav- perfusion chamber (vol, 0.3 ml) with a 0.45-pym metricel mem- ing the same retention as synthetic standard is shown in Table brane filter (Gelman). Each chamber contained the tissue 1. Resolution of replicate extracts gave similar results. equivalent of six hippocampi (200-300 mg of tissue per cham- The total immunoreactivity of each of the peptides was ber) taken from a pool of hippocampal slices. Slices were su- measured in hippocampal extracts (Table 1). Consistent with perfused with modified Krebs-bicarbonate buffer at 0.4 ml/min at 24°C. To determine K+-stimulated release of [3H]norepi- 30 16 30 nephrine (New England Nuclear) from hippocampal slices, slices 112 were incubated in 0.5 ,uM [3H]norepinephrine (10 Ci/mmol; 20 1 Ci = 37 GBq) for 30 min. Perfusate samples were collected 0 =6 in scintillation vials, mixed with Aquasol II (New England Nu- 10 20-B,"I 20 a2) Al -4 -I 3, clear), and radioactivity was determined.
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