Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 3540-3544, May 1991 Medical Sciences

Isolation and characterization of two peptides with prolactin release-inhibiting activity from porcine hypothalami (proopiomelanocortin precursor/neurophysin precursor/prolactin release-inhibiting factor)

ANDREW V. SCHALLY*t, JANOS G. GUOTH*t, TOMMIE W. REDDING*, KATE GROOT*, HENRY RODRIGUEZf, EVA SZONYIt, JOHN STULTS*, AND KAROLY NIKOLICSt *Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center, 1601 Perdido Street, New Orleans, LA 70146; tDepartment of Experimental Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112; and tDepartments of Developmental Biology and Protein Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080

Contributed by Andrew V. Schally, December 28, 1990

Here we report the isolation of two peptides from porcine hypothalami, different from gonadotropin releasing hormone-associated peptide (GAP) and somatostatin-14 (SS-14), that exhibited a dose-dependent prolactin release-inhibiting activity in vitro. These substances or their congeners might play a physiological role in the regulation of prolactin release.

ABSTRACT Two peptides with in vitro prolactin releaseinhibiting activity were purified from stalk median eminence (SME) fragments of 20,000 pig hypothalami. Monolayer cultures ofrat anterior pituitary cells were incubated with aliquots of chromatographic fractions and the inhibition of release of prolactin in vitro was measured by RIA in order to monitor the purification. The hypothalamic tissue extract was separated into 11 fractions by high-performance aqueous size-exclusion chromatography with one fraction showing a 4-fold increase in prolactin release-inhibiting factor (PIF) activity. This material was further purified by semipreparative reversed-phase (RP) HPLC. This process resulted in the separation of two distinct fractions that showed high PIF activity. These were further purified by semipreparative and analytical RP-HPLC to apparent homogeneity as judged by the UV absorbance profiles. Neither of the two peptides showed cross-reactivity with gonadotropin releasing hormone-associated peptide or with somatostatin-14 antibodies. Protein sequence analysis revealed that one of the PIF peptides was Trp-Cys-Leu-Glu-Ser-SerGln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu-AlaCys-Ile-Arg-Ala-Cys-Lys-Pro, identical to residues 27-52 of the N-terminal region of the proopiomelanocortin (POMC) precursor (corresponding to amino acids 1-26 of the 16-kDa fragment). The sequence of the other PIF was Ala-Ser-AspArg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-AlaLeu-Leu-Leu-Arg-Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-GluPro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val-Tyr, representing residues 109-147 of the vasopressin-neurophysin precursor. Synthetic peptides corresponding to the N-terminal region of POMC had significant PIF activity in vitro.

MATERIALS AND METHODS Isolation of the two peptides with PIF activity from 20,000 lyophilized stalk median eminence (SME) fragments of pig hypothalami (Oscar Mayer, Madison, WI) was accomplished essentially by sequential purification in six steps. After each purification step, the fractions were pooled and their in vitro PIF activity and levels of immunoreactive GAP and SS-14 were determined. Extraction. Extraction was carried out as described in detail by Schally et al. (3). Briefly, lyophilized fragments of 20,000 pig hypothalami, weighing 531 g, were first pulverized, defatted by acetone and petroleum ether, extracted with 2 M acetic acid at 80C, and centrifuged (3). Phenylmethylsulfonyl fluoride and pepstatin A (10 pug/ml each) were added to the clear supernatant (14). The mixture was heated to boiling, immediately cooled on ice to 40C, and centrifuged. The clear supernatant was lyophilized, resulting in 114.5 g of dry extract from porcine hypothalami. Preparative Size-Exdusion HPLC. From the lyophilized hypothalamic extract, 98 g (16,200 SME) was dissolved in 50% acetic acid (71.5 ml), diluted with distilled water to 4290 ml (final pH 2), and centrifuged (Sorvall RC S B; 26,890 x g; 30 min). The clear supernatant was then subjected to highperformance aqueous size-exclusion chromatography on a TSK G-2000SW (21.4 x 600 mm) column (Toyo-Soda, Phenomenex, Rancho Palos Verdes, CA), after equilibration of the stationary phase with 10 bed vol of 0.1 M NaCl/0.05 M Tris'HC1, pH 4.4. The flow rate of the mobile phase was 6 ml/min. In total, 88 separate chromatographic runs were performed under identical conditions. In each run, 11 fractions were collected (Fig. la). Preparative Reversed-Phae (RP) HPLC. Fraction JGG2-53 no. 3/1 in aliquots of 90 ml from the TSK G-2000SW column, exhibiting significant in vitro PIF and immunoreactive GAP activities, was subjected to RP chromatography on a Dynamax C18 preparative column (Rainin Woburn, MA) (250 x 41.4 mm; 12-pum particle size; 300-X pore size). A linear gradient was used for the elution of the substances adsorbed to the matrix of the column. Component B of the mobile phase was increased from 0% to 10%o in 70 min

The existence of prolactin release-inhibiting factor(s) (PIF) in rat hypothalamic extracts was first demonstrated by Pasteels (1) and Talwalker et al. (2). Several substances were later identified in mammalian hypothalamic tissues that inhibited the release of prolactin (3-7). In humans, the administration of dopamine receptor antagonists, such as chlorpromazine and other neuroleptics, markedly increases prolactin level, whereas dopamine agonists, such as the ergot derivatives, significantly reduce plasma prolactin concentration (8). Based on these clinical and experimental data, dopamine was considered to be the only physiological PIF. On the other hand, peptidic substances have been partially purified from brain extracts, which also had significant PIF activity (9-12). Our preliminary results also suggested that dopamine is not the only hypothalamic substance with PIF activity (13). However, the isolation of a highly potent peptide PIF from hypothalamic extracts has not yet been accomplished.

Abbreviations: PIF, prolactin release-inhibiting factor; POMC, proopiomelanocortin; GAP, gonadotropin releasing hormone-associated peptide; RP, reversed-phase; SS-14, somatostatin-14; SME, stalk median eminence.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 88 (1991)

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Time, min FIG. 1. Chromatographic purification of the two peptides with prolactin release-inhibitory activity. Extracts of 20,000 porcine hypothalami were chromatographed in successive steps by sizeexclusion HPLC (a) and preparative RP-HPLC (b). The shaded or darkened areas represent fractions with the highest in vitro prolactin release-inhibitory activity that were collected and processed in subsequent steps. Fraction 12 of b (JGG-2-127) was further chromatographed by preparative and analytical RP-HPLC (c-f). Fraction 13 of b was further chromatographed by RP-HPLC (g-i). The shaded areas of f (JGG-7-29-12) and darkened areas of i (JGG-7-169-10), respectively, were analyzed by protein sequencing and mass spectrometry.

(solvent A, 0.1% aqueous trifluoroacetic acid; solvent B, 0.1% aqueous trifluoroacetic acid in 70o acetonitrile), while using a flow rate of 62 ml/min (Fig. lb). Sixteen fractions (JGG-2-127) were collected and pooled from each of the 16 separate runs.

Purification Scheme A. One of the two fractions with the highest in vitro PIF activity (JGG-2-127 no. 12), weighing 100 mg, was dissolved in 50%o solvent A/50% solvent B and filtered through a hydrophilic, chemically resistant, 25-mm nylon Acrodisc filter unit (0.2-pum pore size) (Gelman). It was then subjected to RP chromatography on a Dynamax C18 (Rainin) (250 x 21.4 mm; 12-,um pore size; 300-A particle size) preparative column. The same mobile phase was used as in the previous preparative RP separation step, with a different gradient. Component B of the mobile phase was

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increased from 35% to 70% in 140 min, while using a flow rate of 8 ml/min (Fig. 1c). Twenty-four fractions (JGG-2-149) were collected and Iyophilized. A 16-mg aliquot of the material with the highest in vitro PIF activity (JGG-2-149 no. 14) was further purified on a Vydac C18 (Rainin) 250 X 10.0 mm, 5-pum particle size and 300-A pore size semipreparative RP column, using the same mobile phase as in preparative RP-HPLC. A flow rate of 2 ml/min and a shallow linear gradient was used. Component B of the mobile phase was increased from 40% to 62% in 72 min (Fig. ld). Twenty-five fractions were collected and Iyophilized (JGG-2-189). A 6-mg fraction with the highest PIF activity (JGG-2-189 nos. 17 and 18) was rechromatographed on the same Vydac C18 RP column as in step III using the trifluoroacetic acid/ acetonitrile/water, mobile phase, with a shallow linear gradient, increasing component B of the mobile phase from 35% to 70% in 140 min (Fig. le). The separation resulted in 27 fractions (JGG-7-29). The most potent PIF fraction (JGG-7-29 no. 12), weighing 71 pAg, was subjected to purity tests on an Aquapore RP-300 (Brownlee, Phenomenex), 250 x 1.0 mm, 7-pum, 300-A microbore column, using a linear gradient (solvent A, 0.1% trifluoroacetic acid/water; solvent B, 0.1% trifluoroacetic acid/70% acetonitrile/30%o water) (Fig. if), with, a flow rate of 0.08 ml/min. Component B of the mobile phase was increased from 40% to 52% in 40 min. The fraction was subjected to protein sequencing. Purification Scheme B. The second fraction from preparative RP-HPLC with high in vitro PIF activity (JGG-2-127 no. 13) was dissolved in 50%o solvent A/50% solvent B, filtered through a hydrophilic, chemically resistant, 25-mm nylon Acrodisc filter unit (0.2-pmm pore size) (Gelman) and subjected to repeated separation on the same preparative column and in the same mobile phase as described in the previous RP separation step. However, the linear gradient was designed to be shallow. Component B of the mobile phase was increased from 40% to 62% in 65 min. Sixty fractions were collected and lyophilized (JGG-2-219) (Fig. ig). Fraction JGG-2-219 no. 30 with the highest in vitro PIF activity was further purified on a W-Porex 5C18 (Phenomenex) (250 x 4.6 mm; 5-,um particle size; 300-A pore size) analytical RP column, using the same mobile phase as in preparative RP separation. A flow rate of 1.2 ml/min and a shallow, linear gradient was used. Component B of the mobile phase was increased from 42% to 64% in 40 min. Fifteen fractions were collected and Iyophilized (JGG-7-169) (Fig. lh). The most potent fractions in the PIF bioassay, JGG-7-169 no. 10, weighing 200 pLg, was subjected to a purity test on Aquapore RP-300 under the same conditions as in scheme A (Fig. ii). All the separation procedures were performed at room temperature, but the fractions were collected and kept at 40C. The UV absorbance of the fractions eluted from the columns was measured at 220 and 280 nm. For the preparative purification process, a Beckman HPLC system (Beckman, Berkeley, CA) with a 450 data system controller, two 114 M solvent delivery modules, a 340 organizer, a 165 variable wavelength detector, a Kipp and Zonen BD41 recorder, and a Gilson model 201 fraction collector or a MacRabbit HPLC system (Rainin), with a Knauer UV photometer, a Gilson model 202 fraction collector, a Kipp and Zonen BD41 recorder were used. For the analytical steps, a HP-1090 liquid chromatograph HPLC system (Hewlett-Packard) was used. The solvents were HPLC grade, and the reagents were HPLC or analytical grade in purity, obtained from Burdick and Jackson, Sigma, and from Fluka. Distilled water was also purified through a Milli-Q water purification system (Millipore).

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Prolactin and SS-14 RIA. RIA for prolactin was carried out with materials supplied by the National Hormone and Pituitary Program (National Institute of Diabetes and Digestive and Kidney Diseases). The prolactin concentrations in the samples were measured in duplicate by a double antibody RIA method, using as standard rat prolactin, the RP3 reference preparation. The statistical significance was assessed by Duncan's new multiple range test. For the RIA of SS-14 (15) JH 204 antibody was used. GAP RIA. Substances with GAP immunoreactivity were determined by a RIA method, using the KN-16 antibody in a 1: 40,000 final dilution (16). The iodination of the antigen was performed by the chloramine-T method. The labeled hormone was repurified by gel filtration on a Sephadex G-50 column. The specific activity of the labeled hormone was 1375 ,uCi/,ug (1 Ci = 37 GBq). The standard curve was set up in the range between 0.10 pg/1lt and 1 ng/,pi. The binding of the labeled hormone to the antibody was 28%.' The interassay and intraassay variations were less than 12% and 10%,

respectively. PIF in Vitro Bioassay. The monolayer culture assay will be reported in detail elsewhere. Briefly, anterior pituitaries from donor female rats weighing 200-250 g were removed aseptically and dispersed with 0.3% collagenase in Dispase (50 units/ml) and DNase (10 pug/ml). Cells were washed twice and 0.3 x 106 cells were plated per well in 24-well culture dishes in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal calf serum. Cells were incubated at 370C in 5% C02/95% air for 5 days before they were used in the assay system. On the day of the assay, cultures were washed twice with DMEM without serum. Samples dissolved in this same medium were added as 1-ml aliquots to each of four wells. Cultures were incubated an additional 24 hr and aliquots of the medium were taken after 4 and 24 hr for determination of

prolactin levels by RIA. PIF activity was arbitrarily defined as the prolactin release-inhibiting activity ofthe compound as compared to the effect of the control material in the same bioassay. The effect was expressed as the percentage of activity of the control material in units per SME or units/mg. Since catecholamines and SS-14 contribute to PIF activity in hypothalamic extracts in this assay, the PIF activity in units per SME was at times decreased after a purification step due to the elimination of these substances. Protein Sequencing. Purified fractions of the final chromatographic runs were analyzed by N-terminal protein sequencing. Automated Edman degradation was performed with an Applied Biosystems model 470A gas-phase sequencer equipped with a 120A phenylthiohydantoin amino acid analyzer. Phenylthiohydantoin-derivatized amino acids were identified by RP-HPLC and integrated with a Nelson analytical model 3000 data system. Sequence interpretation was

performed

on a

Vax 11/785 computer (Digital Equip-

ment) as described (17). Electrospray Ionization Mass Spectrometry. Electrospray ionization mass spectra were obtained with a Sciex API III

triple quadrupole mass spectrometer (Thornhill, Ontario, Canada), using a pneumatically assisted Ionspray nebulizer. The peptide was dissolved in 10% formic acid with water/ acetonitrile (1:1), and it was pumped into the nebulizer (5000 V) at 5 1L/min. The orifice was maintained at 120 V. The mass axis was scanned from 600 to 1600 units (u) in 15 sec using 0.2-u steps.

RESULTS The 2 M acetic acid extraction of 20,000 pig hypothalami resulted in a yield of 114.5 g of dry powder. For isolation of fractions with PIF activity, 98 g of this material was subjected to purification by various HPLC methods (Table 1 and Table 2).

Proc. Natl. Acad. Sci. USA 88 (1991) Table 1. HPLC purification scheme for a porcine hypothalamic peptide (JGG-7-29 no. 12) with PIF activity in pituitary cell culture Prolactin Total SS-14, release* GAP, wt pg/SME ng/SME 4 hr 24 hr Sample 20,000 pig 531 g hypothalami AVS-10-122 114.5 g 2 M acetic acid extraction JGG-2-53 no. 3/1 37 74 2.81 JGG-2-127 no. 12 100 mg 12,278 1.86 41 51 JGG-2-149 no. 14 16 mg 1,487 1.24 37 49 JGG-2-189 nos. 17 + 18 1 mg 5,041 18.7 21 33 JGG-7-29 no. 12 131 ,ug 0 0 58 39 The duration of the incubation of the cells with the tested materials was 4 and 24 hr in each experiment. *Decrease in prolactin release, % control.

The TSK preparative size-exclusion HPLC concentrated PIF activity into 11 fractions. JGG-2-53 no. 3/1 was found to be one of the most potent compounds showing 74 units of PIF activity per SME and 576 units of PIF specific activity per mg, in vitro. This heterogenous fraction was concentrated and separated on a preparative C18 reversed-phase column. The process resulted in three fractions with significant PIF activity. The greatest biological activity was found in the 100-mg fraction designated JGG-2-127 no. 12, with 51 units of PIF activity per SME or 2040 units of PIF activity per mg in vitro. The chromatography performed on a C18 column using a shallow gradient (0.25% solvent B increase per min) produced fraction JGG-2-149 no. 14, weighing 16 mg. This fraction showed 49 units of PIF activity per SME or 6125 units of PIF activity per mg and was still heterogenous (Table 1). After purification on another C18 column, two fractions (JGG-2-189 nos. 17 and 18) were obtained (Fig. ld), weighing 1 mg and exhibiting 33 units of PIF activity per SME or 16,500 units of PIF activity per mg. Fraction JGG-2-189 no. 17 exhibited a dose-dependent PIF effect in vitro (Table 1) but showed significant cross-immunoreactivity with SS-14 and GAP in the RIA (Table 1). The final analytical chromatography using acetonitrile/ trifluoroacetic acid/water, mobile phase, with a shallow gradient (0.25% solvent B increase per min) resulted in 71 ,ug of fraction JGG-7-29 no. 12. An estimated 59.5 ,ug of this material was used up for the RIAs and bioassays to guide the purification. Considering all these data, the yield of the HPLC purification of JGG-7-29 no. 12 was 130.5 ,ug or 91.2%. The isolated material exhibited a strong UV absorption at 220 nm and a lesser one at 280 nm and showed 93% homogeneity as assessed by UV absorbance (Fig. 1f) and had a PIF activity estimated at 39 units per SME or 55,715 units/mg. JGG-7-29 no. 12 was found to be the most potent PIF fraction in the 24-hr incubation test of the pituitary cells. This material Table 2. HPLC purification scheme B of a porcine hypothalamic peptide (JGG-7-169 no. 10) with PIF activity Prolactin Total GAP, SS-14, release* wt Sample pg/SME ng/SME 4 hr 24 hr JGG-2-127 no. 13 35 1094 0.65 30 4 JGG-2-219 no. 30 810 5.30 27

JGG-7-169 no. 10 240 ,ug 0 0 13 30 , Not available. Incubation periods of 4 and 24 hr in pituitary cell culture were used for the bioassay. *Decrease in prolactin release, % control.

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exhibited no cross-reactivity in the RIAs for SS-14 or GAP. This chromatographic procedure resulted in a 96.7-fold increase in PIF activity when assayed in the pituitary cell culture system (Table 1). This fraction was subjected to structural analysis. Fraction JGG-2-127 no. 13 with high PIF activity was purified separately according to scheme B (Fig. 1 g-i). The chromatography performed on a Dynamax C18 column but using a shallow gradient (0.25% solvent B increase per min) resulted in JGG-2-219 no. 30 as the most potent fraction. The final semipreparative chromatography, using acetonitrile/ trifluoroacetic acid/water, mobile phase, but shallow gradient (0.25% solvent B increase per min) on a W-Porex C18 column, results in 240 Ag of JGG-7-169 no. 10. An estimated 40 pug of this substance was used for the RIAs and bioassays to guide the purification procedures. Thus, the yield of the HPLC purification of JGG-7-29 no. 12 was 91.2%. The isolated substance exhibited strong UV absorption at 220 nm, no UV absorption at 254 nm, and low absorption at 280 nm, and a PIF activity of 17,391 units/mg. JGG-7-169 no. 10 was found to be a potent fraction showing PIF activity after a 4and a 24-hr incubation period in the pituitary cell culture bioassay. The material showed no cross-reactivity to SS-14 or GAP by RIA. This chromatographic procedure resulted in a 30.1-fold increase in PIF specific activity in the pituitary cell culture system (Table 2). Fraction JGG-7-169 no. 10 was subjected to protein sequence analysis by Edman degradation. Protein Sequencing and Mass Spectrometric Analysis. An aliquot of fraction JGG-7-29 no. 12 was subjected to protein sequencing for 26 cycles. The sample was found to contain as the highest signal Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln-

Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu-Ala-Cys-Ile-ArgAla-Cys-Lys-Pro. The sequence corresponds to residues 27-52 of the N-terminal region of the porcine proopiomelanocortin (POMG) peptide and residues 1-26 of the 16-kDa fragment. Underlying this sequence, another signal was detected in every cycle, which corresponded to the N-terminal region of the a chain of porcine hemoglobin. Another aliquot of this fraction was subjected to electrospray ionization mass spectrometric analysis to determine the size of the fragment. Signals corresponding to six peptides were observed. The identities of these peaks were based on the N-terminal sequences obtained above. A minor peak, Mr 7840.6 + 2.6, corresponds to POMC residues 1-70 (theoretical Mr, 7842.7) and a second peak, Mr 7727.1 ± 6.3, corresponds to residues POMC 1-68 (theoretical Mr, 7728.6). These masses indicate that the four cysteine residues have formed two disulfide bonds and that the N-linked glycosylation site contains no carbohydrate. Major peaks at Mr 7854.6

+ 2.4 and 7740.1 ± 2.3 correspond to POMC residues 1-70 and 1-68, respectively, with a modification, most likely methylation (adds 14 u). Other modifications, such as hydroxylation or methionine oxidation (adds 16 u), although possible, lie at the extremes for expected mass accuracy (±0.02%). One minor peak at Mr 7206.7 + 0.6 corresponded to porcine hemoglobin a chain residues 1-69 (theoretical Mr, 7207.2). Another minor peak ofMr 7452.2 ± 3.8 did not match any sequences of POMC or hemoglobin (assuming the N-terminal sequences found earlier). Fraction JGG-7-169 no. 10 was subjected to protein sequencing. This preparation also gave two main sequences. The dominant sequence was Ala-Ser-Asp-Arg-Ser-Asn-Ala-

Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu-Leu-ArgLeu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-ProAla-Gln-Pro-Gly-Val-Tyr. This sequence is identical to the residues 109-147 peptide region of porcine vasopressinneurophysin, whereas the sequence present in a lesser amount corresponded to an internal region of the porcine hemoglobin a chain.

Proc. Natl. Acad. Sci. USA 88 (1991)

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Table 3. PIF activity of purified hypothalamic PIF and rat, bovine, and human POMC fragments in the pituitary cell culture bioassay Prolactin ng/ml Dose,* releaset At 4 hr At 24 hr 4 hr 24 hr Sample ,ug Control 1730 ± 70 11,413 ± 400 JGG-2-189 no. 17 0.1 1330 ± 60 7,800 ± 600 23f 32t JGG-2-189 no. 17 0.5 1187 ± 170 4,075 ± 600 31§ 64§ Control 1040 ± 22 5,850 ± 150 Rat 0.01 1000 ± 26 4 21§ 4,630 ± 88 0.1 920 ± 19 POMC 3,730 ± 312 11t 36§ 1-49 1.0 790 ± 27 3,230 ± 78 24§ 45§ Bovine 0.01 930 ± 17 4,580 ± 287 11t 22§ 0.1 960 ± 14 POMC 8 15t 4,950 ± 215 1-49 1.0 800 ± 38 4,240 ± 317 23§ 28§ Human 0.01 740 ± 64 4,200 ± 235 29§ 28§ POMC 0.10 920 ± 96 4,280 ± 127 11t 27§ 1-49 1.0 800 ± 10 4,690 ± 120 23§ 20§ *Dry weight. tDecrease in prolactin secretion release, % control. tSignificantly different from control; P < 0.05. §Significantly different from control; P < 0.01.

DISCUSSION The hypothalamic control of pituitary prolactin release is mediated by stimulatory and inhibitory agents (8). Several compounds are known to inhibit prolactin release in pharmacological doses, such as dopamine, norepinephrine, y-aminobutyric acid, and acetylcholine (13). Purification of various fractions with PIF activity from hypothalamic extracts in our laboratory led to the isolation of dopamine and norepinephrine (6), as well as y-aminobutyric acid (7). Dopamine is present in a high concentration in median eminence (18) and in hypophyseal portal vessels (19). Dopamine appears to be a major inhibitory agent in vivo, as it suppresses prolactin levels under a variety of physiological conditions (20). A dopamine agonist, the ergot alkaloid bromocryptine, has been widely used clinically to suppress prolactin in patients with conditions associated with high blood levels of prolactin (8). In addition to catecholamines and y-aminobutyric acid, peptidic PIFs have been demonstrated by various investigators in hypothalamic extracts (9-13). SS-14 was observed to have PIF activity under certain experimental conditions (21, 22). GAP was found to suppress prolactin release in some in vitro tests (16, 23, 24). GAP also had modest prolactin release-inhibiting activity in some in vivo systems associated with high, stimulated prolactin levels (25), but it did not inhibit basal levels of prolactin (26). This peptide was also inactive in the pituitary superfusion system (26). Because of the controversy on possible peptide inhibitors of prolactin release, we attempted to isolate such peptides from hypothalamic extracts by modern separation techniques, monitoring fractions with a sensitive pituitary cell culture assay and determining somatostatin and GAP levels of the fractions by RIA. As a result of this isolation procedure, we purified two fractions with high in vitro prolactin release-inhibiting activity. Both compounds were peptides as based on sequence determination by Edman degradation. One of the peptides was found to be the N-terminal fragment of the POMC precursor protein (27, 28). Synthetic peptides corresponding to this region of POMC-namely, residues 1-28, 1-36 (data not shown), and 1-49-were also found to have significant in vitro PIF activity, comparable to that of the natural molecule (Table 3). However, none of the

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peptides inhibited prolactin secretion in a pituitary superfusion assay (data not shown). It is interesting to note that interleukin 1 (29) and corticotropin-releasing factor (30) have also been found to inhibit prolactin release in vitro. Both molecules stimulate the release of POMC-derived peptides, including corticotropin and f3-endorphin, as well as the N-terminal fragment. It is tempting to speculate that the N-terminal peptide of POMG may be a mediator of the observed effects of interleukin 1. The other peptide obtained from the purification was found to be a fragment (copeptin) of the vasopressin-neurophysin precursor (31). This peptide appeared similar to the glycopeptide previously isolated from pig, ox, and sheep pituitaries (32) and also characterized in other mammalian species (31). Nagy et al. (33) reported that this 39-amino acid glycopeptide comprising the C-terminal of the vasopressin-neurophysin precursor stimulated prolactin release in vitro. The discrepancy between their results (33) and ours can be explained at present only by differences in the assays used. Future in vivo studies can demonstrate whether the substances we isolated, or their congeners, may play a physiological role in prolactin release. We are grateful to Dr. H. P. J. Bennett for the gift of bovine POMC fragments and Dr. R. Acher and Dr. J. Chauvet for sheep MSELneurophysin. The participation in this project of Dr. V. Csernus, Weldon Carter, and Don Olson is gratefully acknowledged. We are grateful to Karl Clauser for his help with mass spectrometric analysis and to Mark Nixon for the synthetic POMC fragment (residues 1-49). We thank the National Hormone and Pituitary Program (National Institute of Diabetes and Digestive and Kidney Diseases) for gifts of materials used in RIAs. This work was supported by National Institutes of Health Grant AM 07467 and the Department of Veterans Affairs Research Service to A.V.S. 1. Pasteels, J. C. (1962) C. R. Acad. Sci. Ser. 2 254, 2664-2666. 2. Talwalker, P. K., Ratner, A. & Meites, J. (1963) Am. J. Physiol. 205, 213-218. 3. Schally, A. V., Kuroshima, A., Ishida, Y., Redding, T. W. & Bowers, C. Y. (1965) Proc. Soc. Exp. Biol. Med. 118, 350-352. 4. Meites, J. & Clemens, J. A. (1972) Vitam. Horm. 30, 165-221. 5. Takahara, J., Arimura, A. & Schally, A. V. (1974) Endocrinology 95, 462-465. 6. Schally, A. V., Dupont, A., Arimura, A., Takahara, J., Redding, T. W., Clemens, R. & Shaar, C. (1976) Acta. Endocrinol. (Copenhagen) 82, 1-14. 7. Schally, A. V., Redding, T. W., Arimura, A., Dupont, A. & Linthicum, G. L. (1977) Endocrinology 100, 681-691. 8. Speroff, L., Glass, R. H. & Kase, N. G. (1983) Clinical Gyne-

9. 10. 11.

12. 13. 14. 15.

16. 17.

18. 19. 20. 21. 22.

23. 24. 25. 26.

27. 28.

29. 30. 31. 32. 33.

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Isolation and characterization of two peptides with prolactin release-inhibiting activity from porcine hypothalami.

Two peptides with in vitro prolactin release-inhibiting activity were purified from stalk median eminence (SME) fragments of 20,000 pig hypothalami. M...
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