Big form of γ-melanotropin-like immunoreactivity in normal human plasma

Peptides.

Vol. 5. pp. 913-916. 1984, " Ankho International Inc. Printed in the U.S.A.

0196-9781/84 $3.00 + .00

Big Form of y-Melanotropin-Like Immunoreactivity in Normal Human Plasma I. T A N A K A , 1 Y. N A K A I , K. N A K A O , T. Y O S H I M A S A S. O K I A N D H. I M U R A

Second Division, Department of Internal Medicine Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan Received 14 July 1983 TANAKA. I.. Y. NAKAI, K. NAKAO, T. YOSH1MASA, S. OKI AND H. IMURA. Big form o/y-melanotropin-like imm,noreactivity in normal human plasma. PEPT1DES 5(5) 913-916, 1984.~Using a radioimmunoassay for Y:c melanotropin (y:~-MSH), y-melanotropin-like immunoreactivity (y-MSH-LI) was detected in plasma extracts of normal subjects before and after metyrapone administration. Plasma y-MSH-L1from four normal men rose significantly after a single oral administration of metyrapone (30 mg/kg of body weight). Gel chromatographic study of plasma extract after metyrapone administration showed a single peak of y-MSH-LI near the elution position of mouse 16K fragment, however smaller forms of y-MSH-LI were not detected. y-Melanotropin

Normalhuman plasma

Metyrapone

y-MELANOTROPIN (y-MSH) is a peptide having amino acid sequence similar to a- and/3-melanotropin, and its presence was first predicted from the nucleotide sequence of DNA complementary to mRNA coding for the precursor [12]. Since a native form of y-MSH was unknown, three y-MSH peptides, y~-MSH (Tyr":'-Val-Met-Gly-His-Phe-ArgTrp-Asp-Arg-Phe4:'-NH2), 3,2-MSH (Tyr~:'-Val-Met-Gly-His Phe-Arg-Trp-Asp-Arg-Phe-Gly4~-OH) and "y:cMSH (Tyr''~Val - Met - Gly - His - Phe - Arg- Trp - Asp - Arg- Phe - Gly - ArgArg-Asn-Giy-Ser- Ser-Ser- Ser-Gly-VaI-Gly-GIy-AIa-AIaGIn""OH), were synthesized [9]. Using synthetic peptides, the action of this putative melanotropin in peripheral tissues and the central nervous system has been investigated [1, 6, 9, 10. 15. 18, 28]. However, the biological role of this peptide has not been elucidated. On the other hand, the presence of y-MSH sequence in adrenocorticotropin (ACTH)-producing tissues was demonstrated by the method of amino acid analysis of extracted peptides [5,20] and radioimmunoassays (RIAs) for synthetic 3,:cMSH [21, 22, 24, 25, 26]. The secretion of y-MSH-like immunoreactivity (y-MSH-LI) from ACTH-producing tumors was also studied in vitro [16,23] and the presence of y-MSH-LI in plasma from patients with Addison's disease, Nelson's syndrome and ectopic ACTHproducing tumor has already been demonstrated [3,14]. However. little is known about y-MSH in normal human plasma. This study was designed to demonstrate the existence of y-MSH-LI in normal human plasma before and after metyrapone administration. RIA for 3,:~-MSHwas used in the present study, because y~-MSH was not found in human pituitary and because glycosylated 3,.~-MSH is considered to be a native form from its structure [27].

METHOD

Preparation of Samples Four normal male volunteers, 30-32 yr old, were studied. The basal blood samples were taken at 9:00 a.m. at the resting state after an overnight fast. All subjects took a single dose of metyrapone (30 mg/kg of body weight, Ciba-Geigy Ltd. Basel, Switzerland) orally at 12:00 p.m. on the same day. The subsequent blood samples were taken at 9:00 a.m. on the next day. All blood samples were withdrawn into chilled siliconized glass tubes containing Trasylol (500 kailkirein inactivator units/ml) and EDTA (1 mg/ml). Plasma was separated by centrifugation at 4°C. An aliquot of plasma was immediately frozen at -20°C for RIAs of ACTH and /3-endorphin and thawed at the time of assay. The remaining portion of plasma was immediately processed for extraction of 3,-MSH.

Extraction of 3,-MSH Plasma y-MSH-LI was extracted by the silicic acid extraction method as described previously [14]. The recoveries of 3,3-MSH added to plasma and 3,-MSH-LI in plasma from patients with Nelson's syndrome and Addison's disease were 70% and 50%, respectively.

Gel Exclusion Chromatography The extracted sample was reconstituted with the standard diluent (0.05 M phosphate buffer, pH 7.4, containing 0.5% human serum albumin (fraction V, ICN Pharmaceutical, Inc., Cleveland, OH), 500 kailikrein inactivator units/ml of

tPresent address: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.

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Trasylol and 0.4% 2-mercaptoethanol) and a 0.3-ml aliquot of reconstituted solution was applied on a Bio-Gel P-60 column (0.7x52 cm), equilibrated and eluted with the standard diluent at 4°C. The descending flow rate was 2.1 ml/hour and the fraction volume was 0.72 ml. Each fraction was subjected to RIA for ya-MSH. The column was calibrated with blue dextran (for void volume), mouse 16K fragment, human ACTH, human fl-endorphin, synthetic y:cMSH and t'-':'I(for the salt peak). Recoveries of mouse 16K fragment, ACTH, 13-endorphin and 7.~-MSH applied on the column were o v e r 90%. RIAs

RIA for 7:cMSH was performed as described previously using synthetic y:cMSH as a reference standard and labelling [24]. The antiserum cross-reacts with 7t-MSH, 72-MSH, mouse 16K fragment and human/3-LPH ( l I%, 5%, 50% and 0.3% on a molar basis, respectively), but not with human ACTH, a - M S H , porcine /3-MSH or human B-endorphin. y - M S H - L I was expressed as tool y.~-MSH equivalents. The minimal detectable plasma concentration of 7-MSH after extraction procedure was 0.7 fmol/ml. RIAs for ACTH and B-endorphin were performed without extraction as described previously [i !,13]. The minimal detectable plasma concentrations of ACTH and/3-endorphin were 6.7 fmol/ml and 3.0 fmoi/ml, respectively.

RESULTS The concentrations of 7-MSH-LI, A C T H - L I and /3-endorphin-LI in plasma before and after metyrapone administration are summarized in Fig. I. The basal concentrations of plasma 7 - M S H - L I in four normal men were 1.4, 1.0, <0.7 and <0.7 fmol/ml, which rose significantly to 4.4-+0.8 fmol/ml (mean-+SE) after oral administration of metyrapone (p<0.01, by Student's t-test). Plasma A C T H - L I and /3-endorphin-LI also increased from the mean basal levels of <6.7 fmol/ml and 10.4__. 1.6 fmol/mi to 29.0-+5.3 fmol/ml and 45.7-+6.6 fmol/ml, respectively, after metyrapone administration (p<0.005, p<0.005). The elution profiles obtained by gel exclusion chromatography of plasma extracts after metyrapone administration are shown in Fig. 2. Only a single peak of 7-MSH-LI which

FIG. 2. Gel filtration profiles on a Bio-Gel P-60 column (0.7>(52 cm) of plasma extracts after metyrapone administration. I--void volume; It--mouse 16K fragment: II1--ACTH: IV--/3-endorphin: V--3,:cMSH; Vl--iodine.

eluted near the elution position of mouse 16K fragment was found in all cases. DISCUSSION We have already observed the presence of 3,-MSH-LI in plasma from two patients with Nelson's syndrome and four patients with Addison's disease, which changed in parallel with plasma A C T H - L I and fl-endorphin-Ll [14]. Gel exclusion chromatography of plasma extracts from these patients revealed a single peak of big ~/-MSH-LI near the elution position of mouse 16K fragment. However y - M S H - L I was not detected in plasma from normal subjects, five patients with Cushing's disease, one of five patients with Addison's disease and one of three patients with Nelson's syndrome, because the minimal detectable concentration of plasma ),-MSH-LI is 51.0 fmol/ml without extraction. Other investigators reported the presence of the amino-terminal portion of the ACTH//3-LPH precursor in normal human plasma using RIAs for the whole amino-terminal portion o f the precursor [8, 17, 19]. In those studies, the processing of ),-MSH-LI was not clear because their antisera were not raised against ~/-MSH. The present study demonstrated that 3,-MSH-LI existed in normal human plasma and rose in parallel with ACTH-L1 and /3-endorphin-Ll after the administration of metyrapone. The plasma concentration of 3,-MSH-LI was approximately 7 times lower than that of A C T H - L I when compared on molar basis. The low concentration of T-MSH-LI, compared to that of ACTH-LI, can be explained in part by the lesser cross-reactivity of big 3~-MSH-LI, since the gel exclusion chromatography of plasma extract after metyrapone administration revealed a single peak of big 7-MSH-LI. This big ~/-MSH-LI eluted near the elution position of mouse 16K fragment and is thought to coincide with N-POC t-¢7. a 77 amino acid glycopeptide of the amino-terminal region of human pituitary ACTH//3-LPH precursor, which was reported to exist in human plasma by Hope e t a / . [81. Although there is a possibility that big 7-MSH-LI may contain more than one component, as 16--17K 3,-MSH and 13-14K 3,-MSH were demonstrated in mouse pituitary tumor cells, AtT-20/DI6v, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [17]. In plasma of rat and horse, small "/-MSH-LI. which is thought to coincide with the glycosylated form of Lys-3,:~-MSH, was

3~-MSH-L1 IN H U M A N P L A S M A

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d e m o n s t r a t e d [19,29[. On the o t h e r hand, small 3,-MSH-LI was not d e m o n s t r a t e d in human plasma in this study. This d i s c r e p a n c y suggests the difference of proteolytic processing of the amino-terminal fragment o f the A C T H / / 3 - L P H precursor in different species. T h e s e results indicate that the native form of 3,-MSH must be taken into c o n s i d e r a t i o n w h e n physiological role of 3,-MSH in peripheral tissues is evaluated.

ACKNOWLEDGEMENTS We are greatly indebted to Drs. R. Guillemin and N. Ling of the Salk Institute, La Jolla, for a gift of synthetic 3,:~-MSH, to Professor C. H. Li. University of California at San Francisco for a gift of human/3-endorphin, to Drs. B. A. Eipper and R. E. Mains, University of Colorado, Denver, for a gift of mouse 16K fragment and to Miss A. Wakai for technical assistance.

REFERENCES 1. AI-Dujaili, E. A. S., B. C. Williams, C. R. W. Edwards, P. Salacinski and P. J. Lowry. Human y-melanotropin precursor potentiates corticotropin-induced adrenal steroidogenesis by stimulating mRNA synthesis. Bh~ehem J 204: 301-305, 1982. 2. Bertagna, X., F. Girard, D. Seurin, J.P. Luton, H. Bricaire, R. E. Mains and B. A. Eipper. Evidence for a peptide similar to 16K fragment in man. Its relationship to ACTH. J Clin Endocrimd Metab 51: 182-184, 1980. 3. Bertagna, X., D. Seurin. L. Pique, J. P. Luton, H. Bricaire and F. Girard. Peptides related to the NHz-terminal end of proopiocortin in man. J Clin Endocrinol Metab 56: 489-495, 1983. 4. Chen, J. S. D., N. G. Seidah and M. Chretien. Measurement of N-terminal (1-76) of human proopiomelanocortin in human plasma: correlation with adrenocorticotropin. J Clin Endocrinol Melah 56: 791-796, 1983. 5. Estivariz, F. E., J. Hope, C. McLean and P. J. Lowry. Purification and characterization of a y-melanotropin precursor from frozen human pituitary glands. Biochem J 191: 125-132, 1980. 6. Farese, R. V., N. C. Ling, H. A. Sabir, R, E. Larson and W. L. Traudeau, 111. Comparison of effects of adrenocorticotropin and Lys-y:~-melanocyte-stimulating hormone on steroidogenesis, adenosine 3', 5'-monophosphate production, and phospholipid metabolism in rat adrenal fasciculata-reticularis cells in vitro. Endocrim~logy 112: 129--132, 1983. 7. Giillner, H. G., W. E, Nichoison, J. R. Gill and D. N. Orth. Plasma immunoreactive proopiomelanocortin-derived peptides in patients with primary hyperaldosteronsm, idiopathic hyperaldosteronism with bilateral adrenal hyperplasia and dexamethasone-suppressible hyperaldosteronism. J C/in Endot'rim~l Metab 56: 853-855. 1983. 8. Hope. J.. S. J. Ratter. F. E. Estivaritz, L. McLoughin and P. J. Lowry. Development of a radioimmunoassay for an aminoterminal peptide of pro-opiocortin containing the a-MSH region: measurement and characterization in human plasma. Clin Eml,ermol 15: 221-227. 1981. 9. Ling. N., S. Ying. S. Minick and R. Guillemin. Synthesis and biological activity of y-melanotropin peptides derived from the cryptic region of the adrenocorticotropin/13-1ipotropin precursor. Lil~" Sci 25: 1773-1780. 1979. 10. Lis. M., P. Hamet. J. Gutkowska, G. Maurice. N. G. Seidah, N. Lariviere. M. Chretien and J. Genest. Effect of N-terminal portion of pro-opiomelanocortin on aldosterone release by human adrenal adenoma in vitro. J Clin Endocrinol Metab 52: 10531056. 1981. I I. Nakai. Y., K. Nakao, S. Oki and H. lmura. Presence of immunoreactive t3-1ipotropin and/3-endorphin in human placenta. Lilt' Sci 23: 2013-2018, 1978. 12. Nakanishi. S.. A. lnoue. T. Kita, M. Nakamura, A. C. Y. Chang. S. N. Cohen and S. Numa. Nucleotide sequence of cloned cDNA for bovine corticotropin-/3-1ipotropin precursor. .~Ollll't" 278: 423--427. 1979.

13. Nakao, K., Y. Nakai, S. Oki, K. Horii and H. lmura. Presence of immunoreactive/3-endorphin in normal human plasma. J Clin Invest 62: 1395-1398, 1978. 14. Nakao, K., S. Oki, I. Tanaka, Y. Nakai and H. lmura. Concomitant secretion of y-MSH with ACTH and /3-endorphin in humans. J Clin Endocrimd Metub 51: 1205-1207, 1980. 15. Oki, S., K. Nakao, Y. Nakai, N. Ling and H. lmura. 'y-MSH' fragment from ACTH//3-LPH precursor have an affinity for opiate receptors. Eur J Pharmacol 64: 161-164, 1980. 16. Oki, S., K. Nakao, 1. Tanaka, K. Horii, Y. Nakai, S. Shimbo, M. Watanabe, T. Nakane, A. Kuwayama, N. Kageyama and H. lmura. Concomitant secretion of adrenocorticotropin,/3-endorphin and y-melanotropin from perfused pituitary tumor cells of Cushing's disease: effects of Lysine vasopressin, rat median eminence extracts, thyrotropin-releasing hormone and luteinizing hormone-releasing hormone. J Clin Endocrinol Metab 52: 42-49, 1981. 17. Oki, S., K. Nakao, 1. Tanaka, F. Kinoshita, Y. Nakai and H. lmura. Characterization of y-melanotropin-like immunoreactivity and its secretion in an adrenocorticotropin-producing mouse pituitary tumor cell line. Endocrinology 111: 418--424, 1982. 18. Pedersen, R. C. and A. C. Brownie. Pro-adrenocorticotropin/ endorphin-derived peptides: coordinate action on adrenal steroidogenesis. Science 208: 1044-1046, 1980. 19. Pedersen, R. C., N. Ling and A. C. Brownie. lmmunoreactive y-melanotropin in rat pituitary and plasma: a partial characterization. Endocrinology I10: 825-834, 1982. 20. Seidah, N. G., S. Benjannet, R. Routhier, G. De Serres, J. Rochemont, M. Lis and M. Chretien. Purification and characterization of the N-terminal fragment of pro-opiomelanocortin from human pituitaries: homology to the bovine sequence. Bioehem Biophys Res Commun 95: 1417-1424, 1980. 21. Shibasaki, T., N. Ling and R. Guillemin. A radioimmunoassay for ~,-melanocyte stimulating hormone. L~fe Sci 26: 1781-1785, 1980. 22. Shibasaki, T., N. Ling and R. Guillemin. Pituitary immunoreactive */-melanotropins are glycosylated oligopeptides. Nature 285: 416-417, 1980. 23. Shibasaki, T. and H. Masui. Effects of various neuropeptides on the secretion of proopiomelanocortin-derived peptides by a cultured pituitary adenoma causing Nelson's syndrome. J Clin Endoerinol Metab 55: 872-876, 1982. 24. Tanaka, I., Y. Nakai, H. Jingami, J. Fukata, K. Nakao, S. Oki, S. Nakanishi, S. Numa and H. Imura. Existence of y-melanotropin (3,-MSH)-Iike irnmunoreactivity in bovine and human pituitary glands. Biochem Biophys Res Commun 94: 211-217, 1980. 25. Tanaka, 1., Y. Nakai, K. Nakao, S. Oki, J. Fukata and H. Imura. y-Melanotropin-like immunoreactivities in human pituitaries, ACTH-producing pituitary adenomas and ectopic ACTH-producing tumours: evidence for an abnormality in glycosylation in ectopic ACTH-producing tumours. Clin Endocrinol 15: 353-361, 1981.

916 26. Tanaka, I., Y. Nakai, K. Nakao, S. Oki, N. Masaki, H. Ohtsuki and H. Imura. Presence of immunoreactive ~/-melanocyte-stimulating hormone, adrenoeorticotropin and ~i-endorphin in human gastric antral mucosa. J Clin Endocrinol Metab 54: 392396, 1982. 27. Tanaka, I., Y. Nakai~ K. Nakao, S. Oki, T. Yoshimasa and H. Imura. yl-Melanotropin-like immunoreactivity in bovine and human adrenocorticotropin-producing tissues. J Clin Endocrinol Metab 56: 1080-1083, 1983.

TANAKA ET AL. 28. van Ree, J. M., B. Bohus, K. M. Csontos. W. H. Gispen. H. M. Greven, F. P. Nijkamp, F. A. Opmeer. G. A. de Roote, T. B. W. Greidanus, A. Witter and D. de Wied. Behavioral profile of ),-MSH: relationship with ACTH and/3-endorphin action. Lil~" Sci 28: 2875-2888, 1981. 29. Wilson, M. G.. W. E. Nicholson, M. A. Holschner, B. J. Sherrell, C. D. Mount and D. N. Orth. Proopiomelanocortin peptides in normal pituitary, pituitary tumor and plasma of normal and Cushing's horses. Endocrinoloey 110: 941-954, 1982.