Content of β-LPH and its fragments (including endorphins) in anterior and intermediate lobes of the bovine pituitary gland

Life Sciences, 9ô1 . 22, pp . 1715-1722 Printed in the II .S .A .

Pergamon Press

CONTENT OF B-LPH AND ITS FRAGMENTS (INCLUDING ENDORPHINS) IN ANTERIOR AND INTERMEDIATE LOBES OF THE BOVINE PITUITARY GLAND Jean-Claude Lissltsky, Odette Morin, AndrB Dupont, Fernand Labrie, N .G . Seidah, Michel Chrétien, Martin Lis and D.H . Coy Medical Research Council Group in Molecular Endocrinology, Le Centre Hospitalier de l'Université Laval, Quebec 61V 4G2, Canada ; Clinical Research Institute, Montreal ; and Endocrine and Polypeptide Laboratories, Tulane University School of Medicine and V.A . Hospital, New Orleans, LA 70146 (Received in final form March 24, 1978)

Summary Radioimmunoassays (RIAs) specific for ß-LPH1~7, ß-endorphin, a.-MSH and ß-FISH have been used to identify immunoreactive components in acid extracts from anterior and intermediate lobes of bovine pituitary gland after separation by chromatography on Sephadex G-50 . When components in extracts of both lobes, eluting at the same position, were measured with the 9-endorphin and ß-LPH' -47 RIA systans, marked quantitative differences were seen . The main components reacting with the ß-LPHI-47 system in anterior pituitary extract co-migrated with ß-LPH and Y-LPH while in the intermediate lobe, the main immunoreactive component eluted at a position slightly later than B-endorphin. When the ß-endorphin RIA system was used, re atlvely low amounts of immunoreactive material co-migrating with ß-endorphin were seen in the anterior lobe extract while a highly predominant peak eluting at a position slightly later than B-endorphin was observed in intermediate lobe extract. Some B-MSH was seen in the intermediate lobe . These date indicate that the processing of ß-LPH is markedly different in the anterior and interniedlate bovine pituitary lobes : B-endorphin immunoreactlve material predominates 1n the intermediate lobe whereas 9-LPH and Y-LPH predominate in the anterior lobe . Soon after elucidation of the structure of the pentapeptide met-enkephalin from porcine brain (1), longer peptides having met-enkephalin at their N-terminus were isolated from pituitary tissue or from the hypothalamo-pituitary complex . These peptides, B-endorphin (2,3), a-endorphin (4) and Y-endorphin (5) bind to the opiate receptor (1, 6-8) and have opiate-like activity in various assays (2-5, 7-17) . Supporting evidence for a role of these endogenous peptides in various brain functions, is rapidly accumulating . In fact, beside their analgesic potency (7, 10, 11) and activity as behavior modulators (12), endogenous opiate-like peptides appear to be involved in the control of prolactin and growth hormone secretion (13-17) . Using immunohistochemical techniques at the light and electron microscope levels, it has been found that not only the same cells but also the same secretory granules in the anterior and intermediate lobes of the pituitary gland of many species contain material reacting with ovine ß-LPH and ACTH antisera (19) . Pituitary glands were also shown to synthesize ß-LPH, Y-LPH and ß-endorphin . (2022) while the intermediate lobe was found to synthesize almost exclusively peptides related to B-LPH (23) . Since the processing of another pituitary peptide, ACTH, has been found to be different in the two pituitary lobes (24), the possibi11ty was raised that the processing of B-LPH might also differ . The interest of such study is strenghtened by the recent report that endorphins and ACTH origi0300-9653/78/0515-1715$02 .00/0 Copyright Q 1978 Pergamon PYess

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pate from the same precursor molecule (25) . As a preliminary step in our investigation, the factors controlling the processing and release of ß-LPH and its fragments (including endorphins) in the anterior and intermediate lobes of the pituitary gland were studied . The present study takes advantage of the availability of specific antisera to ß-LPH, ß-endorphin, a-MSH and ß-MSH as well as the opiate receptor assay combfhed with gel filtration to assess the content and distribution of ß-LPH and its fragments in the two pituitary lobes . Materials and Methods Extraction of anterior and intermediate ituitar lobes . Bovine pituitaries were remove ass than m n fo ow n exsanguinat on of adult animals of unspecified sex in a local slaughterhouse Abattoir St-Charles, Quebec) and immediately carried to the laboratory in ice-cold 0 .9% NaCI . Less than three hours after death of the animals, the anterior and intermediate lobes were then dissected free of the posterior lobe and connective tissue and were homogenized in 5 vol of 2M acetic acid . After centrifugation at 20,000 xg for 20 min, the supernatant was lyophilized . Se hadex G-50 chromato ra h . The lyophilized extract was dissolved in a minima vo ume o . M scat c ac d before gel filtration on Sephadex G-50 (3 .6 x 200 cm) in the same eluent at a flow rate of 0 .2 ml/min . 10 ml fractions were collected, lyophilized and dissolved in 5 ml 25 mM Tris-HC1, 2 mM MgC12 (pH 1 .4) for RIA and measurement of opiate binding activity using C3H]met-enkephalin as described (6) . The markers used were synthetic ß-endorphin and met-enkephalin as well as purified ß o -LPH and Y o -LPH . Antisera . Antibodies to ß o -LPH (26) and ßh-LPH (27) were developed in rabbits . pA~a~M H and beta-MSH antisera also developed in rabbits were generous gifts from Drs . Oliver, Marseille, and Donnadieu, Paris, respectively . ~Pe tides . ß-endorphin, met-enkephalin, a-endorphin and y-endorphin were synthess zi ed by solid-phase methods and purified as described (28, 29) . ß o -LPH was purified as previously described (30) while ß-LPH1_47 was obtained by treatment Synthetic a-MSH and ß-MSH were generous gifts of ß o -LPH with cyanogen bromide . from Ciba-Geigy and NIAMDD, respectively . Radiorece for sees rocedure . The assay was performed with L 3 Hlmet-enkephalin us ng part cu ate tact on from rat brain (6) . ßo -LPHI_47 and ß-endorphin were iodiIodination . Radioimmunoassa rocedure . nate w t actoperox ase {Sigma) . Lactoperoxidase was preincubated during 45 sec by mixing equal volumes of lactoperoxidase (1 u9/u1) and H202 (0 .003%) before transfer of 10 ul to the iodination tubes (polyethylene) containing 1 ug of peptide and 0 .5 mCi of C125I7 Na in 30 ul of the iodination buffer (O .1M sodium phosphate, pH 6 .6) . After 3 min of incubation at room temperature, 0 .4 ml of phosphate buffer (0 .5 M, pH 7 .6) was added and the mixture immediately separated on a 9 x 60 mm column of Sephadex G-50 (superfine) pre-equilibrated with 0 .05M phosphate buffer (pH 7 .6), 0 .1% bovine serum albumin and 0 .1% sodium azide . 1 ml fractions were collected and kept at 2-4 °C . a- and ß-MSH were iodinated with chloramine T and purified on QUSO as described by Oliver (31) . These two iodinated peptides were stored at 2-4o C in O .1N HC1 - 2 .5% human serum albumin . Assa rocedure . All solutions and reagents were prepared in 0 .05M phosphate M NaCI, 0 .25% human serum albumin and 0 .1% sodium azide (pH 7 .6) . u er, Manipulations and incubations were carried out at 2-4°C . Approximately 10,000 cpm of the labelled peptide were added and incubation performed in a final vo-

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lume of 0 .5 ml in 12 x 75 mm glass tubes (except for ß-MSH where polyethylene tubes were used) . After 48 hours of incubation at 2-4oC, 0 .1 ml of pooled bank human plasma was added and separation of'free and bound radioactivity achieved by adding 0 .6 ml of ice-cold 30% polyethylene glycol (W/V, Carbowax 6000, Union Carbide) . After vortexing, tubes were centrifuged at 3000 xg for 15 min and radioactivity in the pellet measured . S ecificit and sensitivit of the a-MSH and ß-MSH RIAs . The characteristics o t e spec f c RI s for a- H an ß- H have been previously described (31, 32) . In addition to this infornmtion, the cross-reactivity of ß o -LPH, ßh-LPH and ß o -LPH~_47 in the ß-MSH RIA are, on a weight basis relative to ß-MSH, 0 .2, 0 .05 and 0 D %, respectively . These peptides show no significant cross-reactivity in the a-MSH RIA . Under the conditions described, significant displacement is achieved with 10 pg/tube of the a- or ß-MSH in the respective assays . ß-LPH~_47 RIA system . The ß-LPH1_47 system used an anti ßo -LPH serum, and ß o This antiserum binds 30% of the tracer at a LPH1_47 as tracer and standard . final dilution of 1 :25,000 and has a lower limit of sensitivity at 50 pg with a usable range from 50 pg to 10 ng and a half-displacement at 200 pg . On a weight basis, the cross-reactivity of this system is 50% for ß o -LPH, 10% for ßh-LPH and less than 0 .1% for a-MSH, ß-MSH, a-endorphin, ß-endorphin, y-endorphin and ACTH and its fragments . ß-LPH61-91 (ß-endorphin) RIA system . The ß-endorphin RIA system used an antibo y ra se aga nst ßh-LPH at a f nal dilution of 1 :30,000, with ßh-endorphin as tracer and standard . At 30% binding of labelled ß o -endorphin, the lower limit of sensitivity of the assay was 25 pg with a useful range up to 5 ng and a half-displacement at 200 pg . The cross-reactivity in this system was 10% for ß o -LPH, 20% for ßh-LPH, 0 .05% for a- and Y-endorphin and less than 0 .01% for ß o -LPH1_47, a-MSH, ß-MSH and ACTN1_24 . Calculations . Radioimmunoassay data were calculated and analyzed with a HewlettPac ar ca culator, model 9930, using a program based on model II of Rodbardand Lewald (33) . Results Fig . lA shows that the main component of the acid extract from anterior pituitary tissue measured with the RIA system specific for the N-terminus of ßLPH (ß-LPH~_~7 system) comigrates with ß-LPH while the second major peak elutes at the pos t on of Y=LPH . A small shoulder can be seen on the low molecular weight side of the ß-LPH marker . A low level of immunoreactivity is also seen in the void volume and fractions 62-66 and 74-78 . The elution pattern is however markedly different for the intermediate lobe extract (Figs 2A and 3A) where the major peak measured with the ß-LPH1_47 system elutes at a size slightly smaller than the ß-endorphin marker . Three peaks of lower amplitude co-migrate with the void volume, ß-LPH and y-LPH markers, while a small peak co-migrates with the ß-endorphin marker . As assessed by the specific ß-MSH RIA, the major component of intermediate lobe extract co-migrated with y-LPH while significant activity eluted at the position of ß-LPH, ß-MSH itself and a fragment of intermediate size between ß LPH and y-LPH (Fig . 3B) . It is thus apparent that many components are visualized by both the ß-LPH1_47 and the ß-MSH RIA systems . When the ß-endorphin RIA system was used, the major components in the anterior pituitary lobe extract migrated with ß-LPH and ß-endorphin itself while two minor components migrating slower than ß-endorphin (fractions 62-66 and 74-

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B-LPR Content in Bovine Pituitary Gland

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Fig . 1 . Chromatography of bovine anterior pituitary extract : A) immunoreactivity in the ß-endorphin (~--~) and the ß-LPHI_47 (o--o) RIAs . B) activity in the opiate radioreceptor assay . 78) could also be seen (Fig . lA) . In the intermediate lobe, as observed previously with the ß-LPH1_4~ system, a markedly different distribution was seen : the major peak eluted at a position slightly slower than s-endorphin while material eluting at the positions of ß-LPH, ß-endorphin and the void volume was present in low amounts (Fig . 2A) . It is interesting to observe that the a-MSH RIA can detect fragments in the intermediate lobe extract which migrate at positions different from those measured with the three above-mentioned RIAs . While the largest peak elutes at the position of a-MSH itself (Figs 2A and 3B), significant amounts of irtmunoreactive material appear in the void volume, at the position of ß-LPH and at a position intermediate between B-LPH and Y-LPH . Major peaks are however found at positions slower and faster than Y-LPH and ß-MSH, respectively . When assayed for their ability to displace C 3 H7met-enkephalin binding from rat brain membranes, three peaks were observed in the anterior (Fig . 1B) and intermediate (Fig . 2B) lobe extracts . These fractions co-migrated with ß-endor phin, a- and y-endorphins and met-enkephalin . Discussion The present data clearly suggest that the processing of ß-LPH is markedly different in the anterior and intermediate pituitary lobes . Using three RIA systems specific for ß-LPH1-47, ß-MSH and ß-endorphin, components of similar size as separated by Sephadex chromatography were seen in the extracts of both lobes . However, marked differences were seen in the relative amounts of each immunoreactive component, thus indicating different activities of the enzymatic

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Fig . 2 . Chromatography of bovine internlediary pituitary extract : A) Immunoreactivity in the ß-endorphin (~--~), ß-LPHl-47 (o--o) and a-MSH (e--e) RIAs . B) Activity in the opiate radioreceptor assay . systems active in each pituitary lobe . Using one RIA system specific for the N-terminus of ß-LPH combined with an opiate receptor assay, it has recently been shown that the profiles of total bovine pituitary and brain extracts were different (34) . In that study, the major endorphin measured by the opiate receptor assay appeared to be ß-endorphin although a minor component of approximately 2000 daltons could also be detected . The present findings of three different peaks co-migrating with ß-endorphin . a- and/or y-endorphin and met-enkephalin could possibly be explained by the different extraction procedures or tracer used . The finding of relatively high levels of Y-LPH in both pituitary lobes suggests that paired basic residues at positions 59 and 60 are the site of preferential cleavage of ß-LPH leading to ß-endorphin and Y-LPH . These relatively high levels of Y-LPH suggest that Y-LPH is stable and/or that the protease responsible for the cleavage of the bond between residues 58 and 59 is highly acThe same relative stability also applies to ß-LPH . tive . It should be mentioned that the antiserum used in the ß-LPHI-47 RIA system has approximately the same affinity for ß-LPH and Y-LPH . Relatively low levels of ß-MSH were found in the intermediate lobe, thus suggesting a slow rate of hydrolysis at the pair of basic residues at positions 39-40 . The possibility of differential rates of release of the different fragments should however be taken into account in the interpretation of pituitary contents . The finding that ß-endorphin is the most potent among the endogenous opiate-like peptides isolated sç far as analgesic (35) and induction of growth hormone and prolactin release l8, 14-18) and the relatively high level of this

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Fig . 3 . Chromatography of bovine intermediary pituitary extract : A) Immunoreactivity in the ß-endor hin (~--~), and ß-LPHl-47 (o--o) RIAS . B) Imnunoreactivity in the ß-MSH (" - ~ and a-MSH (o--p) RIAS . peptide in both pituitary lobes suggests that it could be a source of circulating endorphin of physiological importance . This is supported, at least for the intermediate lobe, by our recent observations of a relatively high rate of ß-endorphin release from these cells in primary culture (R . Vincent, J .C . Lissitszky and F . Labrie, unpublished observations) . We have previously shown that ovine ß-LPH and ACTH antibodies react with cells of the pars intermedia and some scattered cells of the anterior lobe of many species (19) . It is to be expected that in the human and other species, where the intermediate lobe is absent, the secretion of ß-endorphin and ACTH occurs in parallel . This seems to be the case for the secretion of ß-LPH + yLPH and ACTH in the human where parallel changes have been observed in response to stress and during the circadian cycle (36 ) . However, in species which possess independent anterior and intermediate lobes, the opportunity exists for additional mechanisms of specific control of ACTH and ß-LPH fragments (including endorphins) release . Selective release of different peptides originating from the 31K precursor (25) could be achieved by selective proteolysis modulated by specific agents in the two pituitary lobes . Our data showing ß-endorphin immunoreactive material eluting in the void volume and positions of ß-LPH and ß-endorphin in bovine anterior pituitary lobe extracts are in agreement with the recent findings that endorphin antisera re solved three forms of immunoreactive material with apparent molecular weights of 31,000, 11,700 and 3,500 (25) in culture media of the ACTH-secreting mouse pituitary cell line AtT-20/D-16v (25) . The findings of a different elution profile of intermediate lobe pituitary extract indicates that, in analogy with ACTH (24), the processing of ß-LPH is different in the two pituitary lobes . It

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will thus be of great interest to study the activity of the different enzymatic systems responsible for this processing and to gain knowledge about the hypothalamic and/or peripheral factors involved . References 1 . J . HUGHES, T .W . SMITH, H .W . KOSTERLITZ, L .A . FOTHERGILL, B .A . MORGAN and H .R . MORRIS, Nature 258 : 577-579 (1975) . 2 . C .H . LI and CHUNG, Proc . Natl . Acad . Sci . 73 : 1145-1148 (1976) . 3 . A .F . BRADBURY, D .G . SMYTH an C .R . NELL, B ocher . Biophys . Res . Commun . 69 : 950-956 (1976) . 4 . R . GUILLEMIN, N . LING and R . BURGUS, C . R . Acad . Sci . 282 : Série D, 783-785 (1976) . _5 . N . LING, R . BURGUS and R . GUILLEMIN, Proc . Natl . Acad . Sci . 73 : 3942-3946 (1976) . 6 . 0 . MORIN, M .G . CARON, A . DE LEAN and F . LABRIE, Biochem . Biophys . Res . Commun . 73 : 940-946 (1976) . 7 . T.PERT, In Opiates and Opioid Peptides (H . Kosterlitz, S . Archer, E .J . Simon, and A. Goldstein, eds), Elsevier Publishing Co ., Amsterdam . 8 . A . DUPONT, L . CUSAN, 0 . MORIN, G .S . KLEDZIK, D .H . COY, C .H . LI and F . LABRIE, In Current Studies of H pothalamic Function 1978, Part I, Hormones (K . Ledeand W .L . Yeale, eds~, S . Karger Ag, Basel, in press . 9 . A .F . BRADBURY, D .G . SMYTH, C .R . SNELL, J .F .W . DEAKIN and S . WENDLANDT, Biochem . Bio h s . Res . Commun . 74, 748-754 (1977) . 10 .J .D . LLU , N . N , V. RS KY, D . SARANTAKIS, C .D . WISE and L . STIN, Nature 260 : 625-626 (1976) . 11 .A .F . BR~RY, D .G . SMYTH, C .R . SNELL, N .J .M . BIRDSALL and E .D . HULME, Nature 260 : 793-795 (1976) . 12 .F . BLOOM,D . SEGAL, N . LING and R . GUILLEMIN, Science 194 : 630-632 (1976) . 13 .MOTOMATSU, T ., M . LIS, N . SEIDAH and M . CHRETIE~ I,ân .~ Neurol . Sci . 1 : 49-59 (1977) . 14 .A . DUPONT, L . CUSAN, M . GARON, F . LABRIE, and C .H . LI, Proc . Natl . Acad . Sci . USA 74 : 358-359 (1977) . 15 .A . DUP N , L . CUSAN, F . LABRIE, D .H . COY, and C .H . LI, Biochem . Biophys . Res . Commun . 75 : 76-82 (1977) . 16 .F . BRIE, . CUSAN, L . FERLAND, A . DUPONT, C .H . LI, D .H . COY, A . ARIMURA, and A .V . SCHALLY, In "Proc . 10th Congr . Collegium Internat . Neuro-Psychopharmacologium" (P . Deniker, C . Radouco-Thomas and A . Villeneuve, eds), Pergamon Press, Oxford, in press . 17 .L . CUSAN, A . DUPONT, G .S . KLEDZIK, F . LABRIE, D .H . COY, and F . LABRIE, Nature 268 : 544-546 (1977) . 18 .C . RIVI Rte, W . VALE, N . LING, M . BROWN, and R . GUILLEMIN, Endocrinology 100 : 238-241 (1977) . 19 .G . PELLETIER, R . LECLERC, F . LABRIE, J . COTE, M . CHRETIEN b M . LIS, Endocrinolo 100 : 770-776 . 20 .X . E T GN , M . C . GILARDEAU, and M . CHRETIEN, Can . J . Biochem . 52 : 349-356 (1974) . 21 .M . CHRETIEN, M . LIS, C . GILARDEAU, and S . BENJANNET, Can . J . Biochem . 54 : 566-570 (1976) . 22 .P . CRINE, S . BENJANNET, N . SEIDAH, M . LIS, and M . CHRETIEN, Proc . Natl . Acad . Sci . 74, 1403-1406 (1977) . 23 . P . BENJANNET, N . SEIDAH, M . LIS, and M . CHRETIEN, Proc . Natl . Acad . Sci . 74, 4276-4280 (1977) . 24 .A .P . SCOTT, J .G . RATCLIFFE, L .H . REES, J . LANDON, H .P .S . BENNETT, P .J . LOWRY, and C . McMARTIN, Nature 244 : 65-69 (1977) . 25 .R .E . MAINS, B .A . EIPPER, -a~ . LING, Proc . Natl . Acad . Sci . US A 74 : 30143018 (1977) . 26 .R . DESRANLEAU, C . GILARDEAU, and M . CHRETIEN, Endocrinology 91 : 1004-1007 (1972) .

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