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Co-Localization and Characterization of Lmmunoreactive Peptides Derived from Two Opioid Precursors in Guinea Pig Adrenal Glands’

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Co-Localization and Characterization of Lmmunoreactive Peptides Derived from Two Opioid Precursors in Guinea Pig Adrenal Glands’ 0270.6474/85/O% 2-3423$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 5, No. 12, pp. 3423-3427 Prlnted in U.S.A. December 1985 Co-localization and Characterization of lmmunoreactive Peptides Derived from Two Opioid Precursors in Guinea Pig Adrenal Glands’ CHRISTOPHER J. EVANS,* ELIZABETH ERDELYI, JOAN HUNTER, AND JACK D. BARCHAS Nancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305 Abstract Peptides derived from both proenkephalin and prodynor- Human and monkey adrenal glands contain pro-opiomelanocortin- phin have been identified in guinea pig adrenal medulla. In derived peptides in addition to proenkephalin (Nakao et al., 1981; extracts of whole adrenal glands radioimmunoassays di- Evans et al., 1983a), but the specific cellular localization has not rected to the prodynorphin-derived peptides cY-neoendorphin, been established. Although dynorphin (l-1 3)-like immunoreactivity dynorphin A, and dynorphin B detected high concentrations can be detected in bovine adrenal medulla (Day et al., 1982), no of immunoreactive material ranging from 113 to 216 pmol/ other prodynorphin-derived products can be found in this bovine gm. The concentrations measured by radioimmunoassays tissue (Evans et al., 1983b). Guinea pig adrenal is unusual in that it directed to the proenkephalin products met-enkephalin-Arg- is the only species in which we detect prodynorphin as well as Gly-Leu and met-enkephalin-Arg-Phe were 878 and 484 proenkephalin fragments (Evans et al., 1983b). In this study we have pmol/gm, respectively. No metorphamide or dynorphin( 1-8) analyzed the processing products and distribution of the opioid could be detected in the adrenals. Leucine-enkephalin im- precursor products stored in the guinea pig adrenal gland. munoreactivity which can be generated from either prody- norphin or proenkephalin could also be measured in the Materials and Methods extracts. Gel filtration showed the immunoreactive material, All peptldes used In this study were obtained from Peninsula Laboratones with the exception of that measured by the a-neoendorphin (San Carlos, CA). Antisera to the proenkephalin, prodynorphin, and pro- radioimmunoassay, to be predominantly of high molecular opiomelanocortln fragments were generated In rabbits by subcutaneous weight ranging from M, = 3,000 to 12,000. Immunocytochem- injection of thyroglobulin conjugates (details of the procedures used have istry, using well characterized antisera to a-neoendorphin been publlshed (Weber et al., 1982b)). Radioimmunoassays (RIAs) were and met-enkephalin-Arg-Gly-Leu, demonstrated that the pro- performed using lodinated tracer peptides and bound ligand separated from dynorphin and proenkephalin products were present in the unbound using a second antlbody precipitation procedure (Weber et al., same cells in the medulla region of the gland. The results show that two opioid peptide precursors can be localized in TABLE I the same cells and exhibit some common features in their Determination of proenkephalln-, pro-opiomelanocortin-, and processing. As a relatively homogeneous, localized system, prodynorphin-derived peptide concentrations by R/A the guinea pig adrenal gland should prove a valuable, in vivo Acid/acetone extracts were prepared for RIA as described under “Materials model for the study of co-localized opioid precursors. and Methods.” Concentrations are expressed for the average of five whole adrenal glands. The asterisked peptides represent RlAs specifically recogniz- At present there are three known protein precursors that give rise ing single arginine cleavage products based on the bovine proenkephalin to endogenous opioid peptides. These are pro-opiomelanocortin and porcine prodynorphin cDNA sequences. Leu-enkephalin immunoreactiv- (Nakanishi et al., 1979), which contains the sequence of adrenocor- ity, although listed under proenkephalin products, could be generated from ticotropic hormone and P-endorphin, prodynorphin (Kakidani et al., prodynorphin. 1982), and proenkephalin (Comb et al., 1982; Noda et al., 1982). Concentration RIA The opioid peptides generated by intracellular processing of these (PmoVgm) precursors vary considerably with regard to their proteolytic stability, Prodynorphin receptor selectivity, and potency. Although there are considerable a-Neoendorphin (Weber et al., 1982c) 216 + 30.9 species differences, fragments of all three precursor proteins have Dynorphin A (I-17) (Weber et al., 1982a) 129 + 13.2 been shown to’be present in the adrenal gland. All mammals * Dynorphin B 113 + 7.0 investigated contain proenkephalin fragments which by immunocy- * Dynorphln (i-8) (Weber et al., 1982a) 52 tochemistry have been shown to be present in chromaffin cells and Proenkephalin are co-localized and co-secreted with adrenalin (Viveros et al., 1979). Leu-enkephalin 209 f 28.3 Met-enkephalin in RGL 878 f 162 Received February 8, 1985; Revised April 22, 1985; Met-enkephalin RF 484 + 105 Accepted May 23, 1985 * Metorphamide (Weber et al., 1983) 51 ’ This work was supported by National Institute of Mental Health Grant MH Pro-opiomelanocortin P-Endorphin (middle region) (Evans et al., 1983a) 23861. We thank Irene lnman for technical assistance with the radioimmu- ACTH (Evans et al., 1983a) noassays and Sue Poage for preparing the manuscript. a-MSH (Evans et al., 1982) *To whom correspondence should be addressed. 3423 3424 Evans et al. Vol. 5, No. 12, Dec. 1985 MARKER8 A 8 C 0 E ‘I v v v v 8 MET ENK.RF 8 4 Figure I. Gel filtration analysis on Seph- $ adex G75 of the proenkephalin-related im- 0 munoreactrve peptides in guinea pig adrenal z glands. An acid/acetone extract of the G 12 equivalent of one adrenal gland containing $ 10,000 cpm each of the iodinated markers s MET ENK.RGL was loaded onto the column. Aliquots from 5 7 each fraction were analyzed for immuno- reactrve Met-enkephalin-Arg-Phe and Met- E 10 enkephalin-Arg-Gly-Leu. The markers were: 6 A, exclusron; B, ‘251-~-lipotroprn; C, ‘*QI- endorphin; D, ‘?a-neoendorphin; E, ‘25Leu- 5 enkephalrn. a E 8 s 8 4 2 0 I I I 1 I +\ ’ 0 30 40 50 80 70 80 90 FRACTION NUMBER 1982a, b). The properties of the majority of the RlAs used in this study have The assay cross-reacted less than 0.01% with dynorphin(l-8) Met-enkeph- been described and are referenced in Table I. A brief summary of the alin-Arg-Phe, Met-enkephalrn, and a-neoendorphin. The Leu-enkephalin anti- unpublished RlAs is as follows. The Met-enkephalin-Arg-Gly-Leu antisera sera were used at a drlution of 1:50,000 and the lCso ranged between 800 were used at a dilution of 1 :SO,OOO and the I&,, was typrcally 800 to 900 PM. and 1000 PM. The assay cross-reacted less than 0.01% with dynorphrn(l-8) The Journal of Neuroscience Guinea Pig Adrenal-Co-localization of Two Opioid Precursors 3425 MARKERS A B C D E v v v v v 5 OCNEO END 4 3 2 1 0 Figure 2. Analysis of the prodynorphin- immunoreactive products in gurnea pig ad- 3 renals. Preparation of tissue, chromatogra- phy conditions, and iodinated column DYN A markers were Identical to those In Figure 1. Fractions were analyzed using RlAs directed to a-neoendorphin, dynorphin A(l-17), and dynorphln B. 2 1 0 3 DYN B 2 1 0 I I I 1 1 -A\ ’ 0 30 40 50 60 70 80 90 FRACTION NUMBER 3426 Evans et al. Vol. 5, No. 12, Dec. 1985 Tyr-Gly-Gly-Phe, and Met-enkephalrn-Arg-Gly-Leu but had a 2% cross-reac- A t~v~ty wrth Met-enkephalrn. The Met-enkephalrn-Arg-Phe antrsera were used at dilution of 1 3,000, the I&,, rangrng from 1 to 1.5 nM. Met-enkephalrn-Arg- Phe-amide cross-reacted 2% but Met-enkephalrn, Met-enkephalrn-ArgGly- Leu, dynorphrn(l-8) and Met-enkephalrn-Arg-Arg cross-reacted less than 0.1% The dynorphrn B antisera were used at a drlutron of 1:150,000 and had a 1% cross-reactrvrty with dynorphrn B-29 but less than 0.5% cross- reactrvity with a-neoendorphrn, dynorphrn(l-17) and dynorphrn(l-8) The I& was between 200 and 400 PM. Adrenal glands were dissected from male Hartley albino guinea pigs (200 to 300 gm) sacnfrced by decaprtatron. Glands were extracted in 5 w/v of acetone:0.2 N HCI (75 25). Previously we have demonstrated that extractron usrng acid/acetone grves very good recoveries of oprord peptrdes from brarn tissues (from 59 to 80%) (Weber et al., 1982a). Recoveries of svnthetrc ‘251- peptides added to the guinea pig adrenals prior to homogenization was greater than 78% for all peptides to which the assays were developed. The acid/acetone extracts were spun at 10,000 x g for 20 min, the supernatant was evaporated to dryness, and the residue was redissolved in neutral buffer in preparation for RIA. For ael filtration, the residue was dissolved in 25% acetic’ acid and chromatographed in a.0.9 x 120 cm column packed with Sephadex G75 (fine). The column was equilibrated and eluted with 25% acetic acid containing 0.1% thiodiglycol. Fractions of 1.5 ml were collected, and aliquotes were evaporated to dryness and analyzed by RIA For immunohistofluorescence, adrenals were fixed by immersion in 4% formaldehyde at 4°C overnight and then frozen at -8O’C following cryogenic protection in 5% sucrose. Frozen 4.5.pm sections were cut on a Hacker model 5030 microtome and incubated with antisera diluted 1:400. Bound antiserum was visualized with fluorescein-conjugated goat anti-rabbit IgG (see Weber et al., 1982d, for details of the procedure). Blocking controls were performed using 10 FM peptide incubated with the primary antisera. Results and Discussion Whole guinea pig adrenals were extracted with acid/acetone and the concentrations of a series of proenkephalin-, pro-opiomelano- cortin-, and prodynorphin-derived peptides were determined by RIA.
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