BIOSYNTHESIS AND RELEASE OF P-ENDORPHIN-, N-ACETYL p- ENDORPHIN-, /3-ENDORPHIN-(l-27)-, AND N-ACETYL P-ENDORPHIN- (l-27)-LIKE PEPTIDES BY RAT PITUITARY NEUROINTERMEDIATE LOBE: P-ENDORPHIN IS NOT FURTHER PROCESSED BY ANTERIOR LOBE’

Continuous labeling and pulse-chase techniques were employed to study the synthesis and secretion of multiple forms of immunoreactive ,f?-endorphin by cultured dispersed rat anterior lobe cells and intact neurointermediate pituitary lobe. Cell and medium extract immunoreactive pendorphin (specific immunoprecipitation and radioimmunoassay) exhibiting a K,, similar to authentic ,&endorphin upon gel filtration was characterized further by nonequilibrium isoelectric focusing, cation exchange chromatography, reverse phase high pressure liquid chromatography, and partial tryptic and chymotryptic mapping. Intact neurointermediate lobes incorporated radiolabeled amino acids into four to six forms of immunoreactive /3-endorphin. Four of these forms were physicochemically similar to authentic pendorphin, N-acetylated ,L&endorphin, P-endorphin(l-27)) and N-acetylated P-endorphin(l-27). Pulse-chase studies indicated that a /?-lipotropin-like molecule served as a metabolic intermediate for a P-endorphin-like molecule. As P-endorphin-like material accumulated in the cell, some of it was N-acetylated (-18% at 2 hr chase and -65% at 18 hr chase). At later chase times, P-endorphin(l-27)and N-acetylated ,&endorphin-(1-27)-like peptides were the predominant molecular species detected. All endorphin forms were detected in unlabeled tissue maintained in culture or tissue continuously labeled for 72 hr and were released into the medium under basal, stimulatory (lo-@ M norepinephrine), or inhibitory (10e7 M dopamine) incubation conditions. In all cases, P-endorphin(l-27)-like species were the predominant forms (more than 70% of total) present in the cells and released into the medium. In contrast, approximately 90% of radiolabeled immunoreactive /3-endorphin extracted from anterior lobe cells and medium similarly incubated appeared to represent the authentic /3-endorphin molecule. Continuous labeling (72 hr) revealed the p-lipotropin/p-endorphin molar ratio to be approximately 4. We conclude that, in anterior lobe, most of the /3-endorphin is not processed further and is released intact, while in neurointermediate lobe, it serves as a. biosynthetic intermediate. It has now been firmly established that adrenocorticotropic hormone (ACTH)and p-lipotropin-related peptides are derived from a common precursor molecule(s) in the pituitaries of several species thus far studied. Within a species, anterior pituitary corticotropic cells and intermediate lobe cells synthesize identical or very similar common precursor molecules. However, the posttranslational processing of the precursor in the two lobes differs. In rat anterior pituitary (AP), ACTH-(l-39) (and its glycosylated derivative) and /3-lipotropin (/?-LPH) appear as major end products, with lesser amounts of pendorphin (P-EP) present, while in neurointermediate lobe (NIL), relatively little intact ACTH or P-LPH is found. In this lobe, ACTH is processed further to (Ymelanotropin (LU-MSH) and ACTH(18-39) -like peptides. Likewise, ,&LPH is processed further into ,&EPlike and y-LPH-like peptides (see Herbert et al., 1980; Eipper and Mains, 1980; and Chretien et al., 1979, for a review). ’ This work was supported in part by United States Public Health Until recently, P-EP (the 31 COOH-terminal amino Service Grant NB 02893-15 and the Lita Annenberg Hazen Charitable acids of P-lipotropin) was generally thought to represent Trust. We wish to thank H. Houghten for the synthesis of human pa major secretory product in the rat neurointermediate endorphin, ,8-endorphin-(l-27), and their u-N-acetyl derivatives as well lobe, derived from a trypsin-like cleavage of fi-LPH at a as for useful suggestions. pair of basic residues immediately preceding the p-en586 Liotta et al. Vol. 1, No. 6, June 1981 dorphin sequence. Smyth et al. (1978,1979) have isolated four peptides from pig pituitary which they identified as P-EP, ,&EP-(l-27), and their a-N-acetyl derivatives on the basis of chromatographic behavior and amino acid analysis. Very recently, Zakarian and Smyth (1979) identified ,&EP-(1-27) and a-N-acetyl ,f?-EP-(1-27) as the major components of immunoreactive (IR) /?-EP in extracts of freshly obtained rat anterior and intermediate pituitary lobe tissue; authentic P-EP and its a-N-acetyl derivative were minor components. Subsequently, we have confirmed this finding with respect to neurointermediate lobe (Liotta and Krieger, 1980). Since the major species detected exhibit no or only weak opiate agonist activity (Smyth et al., 1979), these findings directly bear on the possible physiological role(s) which these peptides subserve. However, the mere detection of a peptide in a tissue extract cannot be taken as direct evidence that such a peptide represents an actual physiological product. Incomplete inactivation of pituitary enzymes during extraction or postmortem autolysis could produce some of the detected peptides artifactuahy. Furthermore, an intracellular mechanism may exist that, under certain metabolic conditions, degrades peptides otherwise destined for secretion (e.g., lysosomal fusion with secretory vesicles). We therefore sought to determine if the multiple forms of immunoreactive P-endorphin detected in rat pituitary did indeed represent secretory products. We reasoned that, if this were so, (1) their biogenesis should be demonstrable in vitro when pituitary is incubated in the presence of radiolabeled amino acids utilizing pulse-chase and continuous labeling paradigms and (2) they should be released into the medium when tissue is maintained in culture under the appropriate conditions. Materials and Methods Preparation of tissue and conditions of culture. Anterior pituitary cells were prepared as described (Liotta et al., 1979) and plated at 6 to 9 x 10” cells/well. To ensure that no cross-contamination of lobes occurred during dissection, the whole pituitary was removed intact and anterior lobe not in contact with NIL was removed with a pair of iris scissors for dispersion. The remaining anterior tissue adhering to NIL was removed and discarded. Neurointermediate lobe tissue was not dispersed but incubated intact, one per well. Medium 199 supplemented with nonessential amino acids (200 PM), 5% normal rat serum, 5% fetal calf serum, penicillin G (50 units ml-‘), and streptomycin (100 pg ml-‘) was employed. Several batches of serum had to be tested prior to use, since some preparations contained considerable proteolytic activity (added /?-EP and (YMSH were recovered poorly). Anterior pituitary cells and neurointermediate lobes were incubated with 300 ~1 of medium, except for delivery of the 30-min “pulse” of radioactive amino acids, in which case, 100 1.11 was used. When 72-hr prelabeled or unlabeled cells were used to characterize IR-P-EP released into medium (see below), the serum was replaced with 0.8% defatted rat albumin. Incubation of pituitary tissue with radioactive amino acids. Anterior pituitary cells were incubated with [35S]methionine (final specific activity, 23 Ci mmol-’ at a final concentration of 150 pM) continuously for 72 hr. Neurointermediate lobe tissue was similarly incubated with [“5S]methionine and [“Hltyrosine (Tyr; final specific activity, 22 Ci mmol-’ at a final concentration of 200 I*M). For pulse-chase studies, NIL was incubated for 30 min with [35S]methionine at a final specific activity of 100 Ci mmol-’ and a final concentration of 200 pM, after which, the medium was removed and the tissue was washed (1 min/wash) three times with Medium 199 devoid of[35S]methionine (unlabeled methionine was present at 1 mM) and then incubated (chase) in such medium for 1,2, 4, 6, or 18 hr. Release studies. Anterior pituitary cells were incubated for 1 hr with 0.1 equivalent (-80 pg of protein) of a rat medial basal hypothalamic extract (rHME; HE-RP1 obtained from National Institute of Arthritis, Metabolism, and Digestive Diseases). Neurointermediate lobes were incubated 1 or 2 hr with lo-’ M dopamine (DA) or lo-@ M norepinephrine (NE). Then IR-/3-EP released into the medium under these conditions was characterized and compared to IR-/?-EP released when tissue was incubated in medium only (basal release). Subsequent to such incubations, the tissue was extracted and likewise characterized. In addition, total IR-ACTH and total IRcr-MSH was determined on cell and media extracts for anterior cells and NIL tissue, respectively. Radioimmunoassays (RIA). The ACTH and ,&endorphin assays were performed as previously described in our laboratory (Yamaguchi et al., 1980). The ACTH (West) antiserum was obtained from National Institute of Arthritis, Metabolism, and Digestive Diseases. The /3-EP antiserum cross-reacts on an equimolar basis with camel ,&EP, camel ,&EP-( l-27), and the human forms of these peptides and their a-N-acetyl derivatives as well as with the common precursor molecules. It does not react at all with (Yor y-EP. Since the antiserum reacted equally with the human acetylated peptides and since the sequences of rat and camel P-EP are identical (Rubinstein et al., 1977), we assume that all related rat peptides would be recognized likewise. The a-MSH antiserum was a gift from Dr. H. Vaudry (Universite de Rouen, Mont-Saint-Aignan, France). It possesses less than 0.1% cross-reactivity with synthetic porcine or human ACTH. The procedure rtilized was the same used for /3-EP assay. Immunoprectpitation. Antiserum raised to synthetic porcine P-EP was affinity-purified as previously described (Liotta et al., 1979, 1980). This is the same antiserum employed for P-EP radioimmunoassay. Immunoprecipitation was performed as described (Cornstock et al., 1979). Immunoprecipitates were dissolved in 100 ~1 of solution containing 0.1 M HCl, 0.2 M formic acid, and 8 M urea (overnight incubation at room temperature). Gel filtration. Gel filtration was performed on 0.9 x 60 cm columns packed with Sephadex G-50 fine beads as described (Liotta et al., 1979,1980), except that 1% formic acid was included in the eluent. Recoveries from these columns always exceeded 94%. NaDodS04-polyacrylamide gel electrophoresis (SDSPAGE). This procedure was employed as previously described (Liotta et al., 1980). Recovery of applied radioactivity always exceeded 93% Concentration and desalting of samples. C-18 SepThe Journal of Neuroscience Synthesis of Multiple Forms of Immunoreactive /3-Endorphin 587 Pak cartridges (Waters Associates) were utilized. For elution of peptides, 80% CH&N in 0.05 M HCl or 0.06 M HzS04 was employed. Cation exchange chromatography. IR-P-EP obtained from the gel filtration studies was applied to a 0.9 x 15 cm column of SP-Sephadex equilibrated with 0.04 M HCl. A linear gradient of NaCl in 0.04 M HCl was established utilizing a three-channel peristaltic pump and a mixer volume of 100 ml. The flow rate to the column was 0.25 ml min-‘; 0.75 M NaCl was pumped to the mixer vessel at 0.125 ml mini’ and l-ml fractions were collected. In order to avoid any carry-over of peptides, these columns were used only once. Recovery from these columns always exceeded 90%. Nonequilibrium isoelectric focusing. We define nonequilibrium isoelectric focusing as an electrophoretic procedure utilizing conventional isoelectric focusing equipment and reagents but terminating the run before the molecular species under study have migrated to their respective isoelectric points. This procedure was used rather than isoelectric focusing due to the very basic isoelectric points of &EP and ,&-EP-( l-27). Under equilibrium conditions, these peptides cannot be resolved, since they migrate to the cathodal end of the focusing gel. Since the pIs of pr-EP-( l-27) and its cr-N-acetyl form should fall somewhere between the pK, of the a-NH2 and the pK, of the e-NH2 of lysine and the pK, of the histidine imidazolium function, respectively, termination of the focusing run prior to reaching the neutral region of the gel results in a greater separation of these peptides. A Bio-Rad model 150A vertical tube gel apparatus and IO-cm 7.5% acrylamide gels containing 2% Bio-Lytes, pH range 3 to 10, were employed. Samples were dissolved in 25 to 100 ~1 of 0.06 N HzS04 containing 1% Bio-Lytes and 8 M urea. Samples were applied to the anode side of the gel and focused for 2.5 hr with a limiting voltage of 200 V. The pH gradient of each gel was determined using a surface electrode (Bio-Rad Laboratories). Gels were sliced at 2-mm intervals and eluted overnight at 10°C with 0.5 to 1 ml of a solution containing 0.25% Triton X100, 0.8% human serum albumin, 0.1 M Na2HP04 and 22 mM Na2EDTA, pH 7.5. Marker peptides were run on separate gels. These included camel ,B-endorphin, camel P-EP-( l-27), and the human forms of /?-EP, P-EP-( l-27) and their cr-N-acetylated derivatives. The human peptides were run for comparative purposes only, since they possess lower charge-to-mass ratios than their rat analogues. Recoveries from the gels always exceeded 96%. High pressure liquid chromatography. Reverse phase high pressure liquid chromatography (HPLC) was performed utilizing a Beckman model 332 two-pump, microprocessor-controlled apparatus and a 0.46 x 25 cm Altex ODS column (C-18; 5 FM). Two solvent systems were employed: (1) 0.05 M formic acid/triethylamine (0.25 M formic acid stock solution adjusted to pH 3.0 with triethylamine) using a gradient of acetonitrile and (2) 0.5 M formic acid, 0.14 M pyridine using 1-propanol as the organic modifier (Lewis et al., 1979). Prior to injection, all samples were incubated at 45°C in the presence of 1 M dithiothreitol (DTT) (Houghten and Li, 1979; R. A. Houghten, personal communication). Recovery of IR-P-EP always exceeded 87%. Trypsinization of radiolabeled IR-P-endorphin-(l-9). Sample was dissolved in 200 ~1 of 0.1 M sodium phosphate, pH 7.8, containing 50 pg of human serum albumin. Ten micrograms of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)/trypsin was added and incubated for 16 to 20 hr at 37°C. Chymotrypsinization of radiolabeled IR-P-endorphin-(l-9). The same procedure was employed as for trypsinization. Source of syntheticpeptides. Synthetic camel ,&endorphin (P,.-EP) and synthetic &EP-(l-27) were obtained from Peninsula Laboratories and Bio-Flex Laboratories, respectively. Dr. R. A. Houghten (Division of Endocrinology, Mount Sinai School of Medicine) kindly provided the following synthetic peptides: P-EP-( l-9), human [“H]Tyr’ e/3-EP, human ,&EP, /?-EP-(l-27), and their a-N-acetyl derivatives. /3-[“H]EP-(l-4) was obtained by chymotrypsinization of [“H]Tyr’./?-EP in the presence of 1 mg of unlabeled ,&-endorphin; it was purified by HPLC and its identity was confirmed by amino acid analysis and by liberation of [“H]Tyr with aminopeptidase M. a-N-Acetyl P-EP-(l-9) was prepared by tryptic digestion of Q-Nacetyl ,&-EP; its amino acid sequence was confirmed by amino acid analysis (Bio-Flex Laboratories) and the presence of the acetyl group was confirmed indirectly by its resistance of aminopeptidase M (as suggested by D. G. Smyth, National Institute for Medical Research, The Ridgeway, Mill Hill, London). P-EP-(5-9) was obtained by chymotrypsinization of P-EP(I-9) and its sequence was confirmed by microsequencing. Analysis of immunoreactive P-endorphin. Since the purpose of this study was to characterize forms of IR-PEP with the approximate molecular size of the authentic /?-EP molecule, all tissue and medium extracts were submitted to molecular sieve chromatography (Sephadex gel filtration) and material eluting in the region (with a similar K,,) of the synthetic camel ,&EP marker was pooled, lyophilized, and reconstituted in the appropriate vehicle, and aliquots were analyzed further by nonequilibrium isoelectric focusing, SP-Sephadex cation exchange chromatography, and reverse phase HPLC. Synthetic camel P-EP and /3-EP-( l-27) were used as marker peptides in all separation systems. The a-N-acetyl derivatives were not available to us, but their expected behavior could be estimated on theoretical grounds.” IR-/?-EP radiolabeled during the 72-hr incubation of tissue in medium containing [““Slmethionine and [“HItyrosine was analyzed and purified according to the scheme depicted in Figure 1. The radiolabeled IR-/3-EP forms obtained by this purification procedure also were subjected to trypsinization. The ,&EP-( 1-9)-like peptides generated were identified by (1) HPLC analysis, (2) immunoprecipitation with a methionine enkephalin antibody (Liotta and Krieger, 1979) that recognizes P-EP(l-9) but no other tryptic fragment of P-EP, (3) detection ‘For instance, the acetylated peptides possess one less positive charge and should migrate more slowly than the corresponding unacetylated peptides in the electrophoretic procedure, elute just before them in the cation exchange chromatcgraphic system, and be retained longer in the reverse phase HPLC system. 588 Liotta et al. Vol. 1, No. 6, June 1981