Presence of met-enkephalin in a somatostatin-synthesizing cell line derived from the fetal mouse hypothalamus

Neuropeptides 2: 351-369, 1982

PRESENCE OF MET-ENKEPHALIN IN A SOMATOSTATIN-SYNTHESIZING CELL LINE DERIVED FROM THE FETAL MOUSE HYPOTHALAMUS

F. Cesselin, M. Hamon, S. Bourffoin, N. Buissonx and De Vitry,-F.X.

Groupe NB, INSERM U Cellulaire, College requests to F.C.).

114 and f Groupe de Neuroendocrinologie de France, Paris, France (reprint

ABSTRACT

Immunocytochemical observations with purified antibodies revealed the presence ofmet-enkephalin-like material in the primitive nerve cell line F7 derived from the fetal mouse hypothalamus and previously shown to synthesize somatostatin (1). Radioimmunoassays associated with Bio-gel P2 chromatography confirmed that met-enkephalin itself accounted (at least partly) for the positive immunocytochemical reaction. Trypsinization (+ carboxypeptidase B treatment) of cell extracts significantly i&reased their met-enkephalin-like immunoreactivity therefore suggesting that met-enkephalin precursors were also present in the F7 clone. Parallel studies on the hypothalamus of fetal mice indicated that met-enkephalin but apparently no precursor was already present at embryonic day 15.

The clonal cells F7 may be an appropriate model for investi- gating the functional correlate of the co-occurrence of met- enkephalin and somatostatin in the same cells.

INTRODUCTION

Since the discovery of met-and leu-enkephalins by Hughes et al. (2) in the CNS, several convergent observations revealed that these peptides are often associated with another neurotrans- mitter in the same cells (see 3 for a review). This is notably the case for some raphe neurones containing both serotonin and leu-enkephalin (4) and for chromaffin cells of adrenal medulla (5) and a neuroblastoma clone (NlE-115) (6) which contain enkephalins and catecholamines. In addition, the other neurotrans- mitter can be another peptide such as oxytocin with met-enkephalin or vasopressin with leu-enkephalin in hypothalamic neurones projecting into the neurohypophysis (7). Similarly, substance P and leu-enkephalin have been shown to co-occur

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within numerous neurones of widespread regions of the avian CNS (8). Among such associations, that of met-and/or leu-enkephalin(s) with somatostatin has been found in several cell types including pheochromocytomas (9) and hypothalamic neurones (10,ll).

In most cases, the functional correlate of the coexistence of two (or more) neurotransmitters in the same cell is largely unknown, particularly in brain. One possible way to analyse the respective roles of the two neurotransmitters may consist of using tissue preparations much less complex than the CNS, particularly cultured clonal cells. In this respect, recent investi- gations onthe murine neuroblastoma clone NlE-115 (12) largely contributed to the present knowledge of the functional relation- ships between catecholamines and met-enkephalin in those cells. This prompted us to look for other clones having the capacity to synthesize at least two neurotransmitters.In this paper, we describe the presence of met-enkephalin in a primitive nerve cell line (F7 clone) previously shown to synthesize somatostatin (1). This clone should be of great help to explore further the functional correlate hypothesized by Tramu et al. (11) about the coexistence of somatostatin and enkephalins within the same neurones.

In addition, since the F7 clone was obtained by Sv40 trans- formation of mouse fetal hypothalamic cells (13), the status of met-enkephalin was also investigated in the hypothalamus of mouse embryos. The present data reveal that met-enkephalin can be detected in the mouse hypothalamus already on the 15th day of embryonic development.

MATERIAL AND METHODS

The following compounds were used : cytochrome C (Calbiochem), lysozyme (Sigma), trypsin TPCK (Worthington), carboxypeptidase B (Sigma), CH-Sepharose-4B (Pharmacia), SO-met-enkephalin (Bachem), met-and leu-enkephalins (Peninsula), met-enkephalin-lys6 (Peninsula), met-enke halin-arg6 (Bachem), met-enkephalin-arg6r Phe7 (Bachem), human P -endorphin (Beckman). Other peptides (di, tri and tetrapeptides related to enkephalins) were generously given by Dr M.C. Fournie-Zaluski (Faculte de Pharmacie, Paris, France). The labelled ligand used for radioimmunoassays, (125I)- met-enkephalin (2OOOCi/mmol), was obtained as previously described (14).

Pregnant mice (A/J strain) were kept in a controlled envi- ronment (24"C, 60% relative humidity, alternate cycles of 12h light and 12h darkness, food and water ad libitum) for their whole life. They were killed by decapitation and their embryos were rapidly removed and placed on the stage of a binocular microscope. Immediately after dissection, the hypothalamus was sonicated in O.lN HCl (l:lO, w/v) and heated for 10 min at 95OC. After centrifugation, the supernatant was used as the starting extract for met-enkephalin radioimmunoassay.

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Cell culture

F7 hypothalamic cell line was grown in Falcon plastic tissue culture bottles containing the following medium : Ham FlO (Gibco) plus 15% heat inactivated horse serum (IBF, France), 2.5% fetal calf serum (IBF),1.5% of 200 mM L-glutamine, penicil- lin (50 IU/ml) and streptomycin (50 pg/ml). The bottles were incubated at 37'C at saturating humidity in 10% CO2-90% air atmosphere (see ref. 1 for details).

Preparation of met-enkephalin antibodies

Immunogenic conjugates of met-enkephalin were prepared by two different procedures : (I)-by coupling the peptide to haemocyanin (Sigma) with glutaraldehyde (15) or (II)-to ovalbumin with l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (14). Each reaction was carried out in the presence of 0.1 PCi of 3H-met-enkephalin (The Radiochemicalcentre, Amersham, 31 Ci/ amol). After extensive dialysis treatment against 0.9% NaCl, the conjugates were estimated to contain (I) : 65 met-enkephalin residues per molecule of haemocyanin and (II) : 6 met-enkephalin residues per molecule of ovalbumin. Aliquots (corresponding to 1.5 mg of each conjugate)were emulsified in complete Freund's adjuvant and injected intradermally into the axillar and crural regions of white male rabbits. They were bled 3 weeks later and repea- tedly boosted with 0.75 mg of antigen conjugate (I) or (II) approximately every month. Collected sera were heated (30 min at 56'C) to inactivate the peptidases. They were then mixed with an equal volume of glyceroland kept at -3O'C. Results presented in this paper were obtained with sera collected one month after the 5th booster injections. Immunocytochemistry was performed with antibodies from serum I given by a rabbit injected with conjugate I; serum II corresponding to conjugate II was used for radioimmunoassays.

Met-enkephalin antibodies in serum I were further purified by affinity chromatography using a met-enkephalin-CH Sepharose 4B column. The coupling of met-enkephalin to the activated CH Sepharose 4B was achieved by mixing 7 mg of met-enkephalin with 350 mg of activated CH Sepharose 4B in O.lM C03HNa containing 0.5M NaCl. After a gentle agitation for 3 hours at 25"C, the gel was washed with O.lM C03HNa containing 0.5M NaCl and the remaining free active radicals were blocked with ethanolamine (1M) at pH 9. Finally, the gel was washed withO.lM Tris-HCl, pH 8.0, containing 1M NaCl and kept at 4'C in 0.05M Tris-HCl, PH 7,4, supplemented with 0.01% sodium azide.

Glycerol in the complete serum was removed by extensive dialysis against 0.9% NaCl and the globulin fraction was precipitated with ammonium sulfate at half saturation. After centrifugation , the pellet was dissolved in 0.025M Tris-HCl, pH 7.6, and passed through a small column of Sephadex G25 (PDlO, Pharmacia) to remove the remaining ammonium sulfate. The Sephadex

353

eluate was then poured onto a column of met-enkephalin-CH Sepharose 4B (0.8 cm in diameter; 2cm h). The effluent was collected at O'C and the column was washed with 10 ml of 0.025M Tris-HCl, pH 7.6, and then eluted using 10 ml of 0.2M glycine- HCl, pH 2.6,and 10 ml of O.lN CH3 COOH, pH 3.0, successively. Eluates were collected at 0“C and immediately adjusted to pH 7.6 with 1N NaOH. Column effluent, washing and eluates were then lyophilized, dissolved in 0.025M Tris-HCl, pH 7.6, and passed through small Sephadex G25 columns (as above) to remove salts in excess. The respective Sephadex eluates were then mixed with equal volumes of glycerol and kept at -3O'C.

The various fractions obtained were then checked for their met-enkephalin binding capacities using a modification of the radioimmunoassay described previously (16). Briefly, 0.05 ml of various dilutions of the whole antiserum, 50% S ammonium sulfate precipitate and fractions obtained from the met-enkephalin-CH Sepharose 4B chromatography were mixed with 0.2 ml of 0.025 M Tris-HCl, pH 7.6,containing 5 mg/ml of lysozyme and 0.05 ml of (1251)-met-enke halin at 4OC, the (12E

(3,500 cpm). After an incubation for 20h I)-met-enkephalin-antibodies complex were preci-

Bp;z$"Tl';S a mixture of polyethyleneglycol and gamma-globulins. I)-met-enkephalin was then estimated by radioactivity

counting in a 5110 Packard gamma spectrometer.

As illustrated in fig. 1, no significant met-enkephalin binding capacity was detected in the effluent of the met-enkephalin-CH Sepharose 4B column. Although some elution of met-enkephalin antibodies occurred with 0.2M Gly-HCl, most of the specific antibodies came off the gel with O.lN CH3COOH . Indeed, the met- enkephalin-binding capacity expressed per ng protein was about 5 and 8 times as high in the CH~COOH eluate as in the globulin fraction (precipitating at half saturated ammonium sulfate) and the complete serum respectively (fig.1). Purified antibodies were quite specific since leu-enkephalin and j3-endorphin were very poorly recognized : 0.4% and 1.7% of cross reactivity as compared to met-enkephalin.However met-enkephalin-sulfoxide (SO-met-enkephalin) was a better ligand than met-enkephalin itself (200% of cross reactivity).

Immunocytochemistry

Met-enkephalin was detected by means of the indirect peroxidase labelled antibody technique (17). Cells grown in plastic bottles were fixed for 2 hours with cold 8% formaldehyde in O.lM Na phosphate buffer, pH 7.4, and then rinsed with the same buffer supplemented with 0.9% NaCl. They were subsequently exposed for 24-48 hours at 4OC to purified anti-met-enkephalin antibodies (1:20 final dilution of the CH3COOH eluate), washed for one hour with Na phosphate buffer and incubated with sheep anti-rabbit globulin coupled with horseradish peroxidase (Institut Pasteur : l/100 final dilution). After washing, cells were incubated for 10 min in O.lM Tris-HCl, pH 7.6, containing 0.02% H202

354

and 3,3'-diaminobenzidine (0.3 mg/ml; Sigma). They were rinsed with Tris-HCl buffer and finally mounted in glycerol/gelatin (Sigma). The specificity of the reaction was tested by using preimmune rabbit serum, purified antibodies previously absorbed by met-enkephalin (50 1-19 per ml of the CH3COOH eluate) or the effluent of the met-enkephalin-CH Sepharose 4B column.

Trypsin and carboxypeptidase B treatments

After extensive rinsing with O.lM Na phosphate buffer, pH 7.4, containing 0.98 NaCl, cells were scrapped off the bottles and pelleted by centrifugation. They were sonicated in O.lN HCl, immediately heated at 95°C for 10 min and finally centrifuged (9,500 g for 10 min at 4OC). Cell and hypothalamic supernatants were adjusted to pH 8.6 with 1M Tris and then mixed with an equal volume of a trypsin (TPCX) solution in 0.05M Tris-HCl, pH 8.6. The concentration of trypsin in the final mixtures was equal to 0.25 mg/ml (containing 1 mg protein). After an incubation at 37°C for 16h, the reaction was stopped by adding HCl (O.lN final concentration) at-d heating(10 min at 95'C). The assay mixtures were adjusted to pH 7.6 with 1M Tris and centrifuged (9,500 g, 10 min, 4'C). The resulting supernatants were directly used for the radioimmunoassay of met-enkephalin. When a second treatment with carboxypeptidase B was carried out, trypsinization was stopped only by heating (95'C for 10 min). After cooling to 37'c, carboxypeptidase B (30 yg/ml) was added and the raction proceeded for a further two hours. Finally the reaction was stopped using the same procedure as after trypsinization alone.

Fig 1 : Met-enkephalin-binding capacities of various fractions extracted from the whole anti-met-enkephalin rabbit serum I

The rabbit antiserum I was fractionated using ammonium sulfate precipitation and affinity chromatography onto a met-enkephalin- CH Sepharose 4B column as described in "Material and Methods". Aliquots of each fraction were then incubated for 20 h at 4'C with (1251) -metienkephalin and the bound radioactivity was precipitated with y-globulins and polyethylene glycol (16). Binding capacity is expressed as percent of total radioactivity bound (ordinate) per E-19 of protein (abscissa) in each fraction. Each point is the mean of triplicate determinations. The highest binding capacity was found in the CH3COOH eluate (0) of the met-enkephalin-CH Sepharose 4B column; on a per pg protein basis, to ).

it was 8 times higher than that of the whole antiserum

Radioimmunoassay of met-enkephalin

The radioimmunoassay of met-enkephalin was carried out as previously described (16). Briefly, an aliquot (0.05-0.1 ml) of each cell or hypothalamic extract adjusted to pH 7.6 was mixed with 0.15-0.10 ml of 0.025M Tris-HCl, pH 7.6, containing 5 mg/ml of lysozyme, and 0.05 ml of an antiserum (II) dilution (l/2,600

(12 in the same buffer. After an incubation for 48h at

4Oc, 4 I)-met-enkephalin (3-4,000 cpm in 0.05 ml) was added and the incubation proceeded for a further 8h at 4°C. Finally, the (1251)-met-enkephalin bound to antibodies was precipitated and counted. Under these conditions, as litte as 0.1 pg of met- enkephalin was detected in 0.05 ml of a given sample (see fig.3). Extensive studies of the cross reactivities of various peptides indicated that neither leu-enkephalin, beta-endorphin, nor related peptides were significantly recognized by the antiserum II (Table 1). However, in addition to met-enkephalj met-enkephalin- arg6r met-enkephalin-arg6-phe7, SO-met-enkephalin-arg6 and particularly SO-met-enkephalin were found to bind to antiserum II. Met-enkephalin-lys6 exhibited only a discrete cross reactivity (Table 1).

356

Table 1

Tested Compound % cross reactivity

Tyr-gly-gly-phe-met-SO Tyr-gly-gly-phe-met-SO-arg6 Tyr-gly-gLyzphe-met Tyr-gly-gly-phe-met-lys6 Tyr-gly-gly-phe-met-arg6 Tyr-gly-gly-phe-met-arg6-phe7 Tyr-gly-gly-phe-leu Tyr-gly-gly-phe Tyr-gly-gly Tyr Tyr-tyr

gly-gly-phe-met gly-gly-phe-leu gly-gly-phe

gly-phe-met

Human p-endorphin

360 21

1s 2.9

28.6 19

<El <O.Ol <O.Ol <O.Ol 0.3 0.03 (0.01 <O.Ol

0.2

Table 1 : Cross reactivity of various compounds relative to met-enkephalin with the rabbit antiserum II

Cross reactivity was calculated (on a molar basis) at 50% displacement according to Abraham (18) whenever possible. Otherwise, it was estimated from the largest amount of a tested compound, i.e. 100 ng.

Identification of met-enkephalin

Bio-gel P2 chromatography was performed at 4'C with columns of 54 cm height and 0.9 cm in diameter using 0.2 N acetic acid as the eluent. The void volume of the gel (19 ml) was determined with cytochrome C. The collected fractions (0.9 ml corresponding to 15 min) were lyophilized and the dry residues were dissolved in 0.2 - 0.5 ml of 0.025 M Tris-HCl, pH 7.6, containing 5 mg/ml of lysozyme. Aliquots (0.1 ml) were used for the radioimmunoassay of met-enkephalin in duplicate.

Proteins were measured accordingto the Olin-phenol procedure (19) with bovine serum albumin (Sigma) as the standard.

Statistical calculations were performed as described by Snedecor and Cochran (20). When the P value (Student's t test) was higher than 0.05,a difference was not considered to be significant.

357

RESULTS

lnocytochemical detection of met-enkephalin-like material in !lonal cell line

Fis. 2A

Fig. 2B

35%

Fig2 : Immunocytochemical detection of met-enkephalin-like material in F7 clonal cells

A. Immunoperoxidase staining with purified anti-met-enkephalin antibodies. The staining is often concentrated at one pole of the cytoplasm (x 500).

B. Control staining with the effluent of the met-enkephalin- CH-Sepharose 4B colurz~ Note the absence of staining (x 500).

Cells of clone F7 presented a positive reaction with either complete antiserum I or purified antibodies (fig. 2A). Although the degree of positive reaction varied from one cell to another, the coloured product was generally concentrated at one pole of the cytoplasm, near the nucleus which remained uncoloured (Fig. 2A). When the effluent of the met-enkephalin-CH-Sepharose 4B column was used instead of the purified antibodies, no staining was seen (Fig. 2B). Similar negative results were obtained with purified antibodies previously absorbed by met-enkephalin or with the preimmune serum.

Immunochemical detection of met-enkephalin-like material in the F7 clonal cells and in the hypothalamus of fetal mice

Met-enkephalin-like material (pg/mg protein)

Clonal cells F7

Experiment no 1 2 3 4 5

62.9 30.5 69.8 9.6 12.5

- m = 37.1 + 12.5 -

Fetal hypothalamus

ED 15 313.2 + 23.0

ED 18 790.5 i- 69.1 -

Table 2 : Levels of met-enkephalin-like material in clonal cells F7 and in fetal mouse hypothalamus at two different embryonic ages (15 and 18 days)

Values are expressed as pg met-enkephalin equivalents per mg protein. For each experiment (l-51, the given value is the mean of 4 series of duplicate determinations made on 4 different aliquots of the cell extract. The levels in fetal hypothalamus are the means + SEM of at least 4 separate determinations.

359

As shown in table 2, the presence of met-enkephalin-like material in F7 clonal cells was confirmed by radioimmunoassay with antiserum II. The nature of the culture medium apparently influenced the cell content of immunoreactive materal since quite different values were found in the two sets of experiments l-3 and 4-5 which corresponded to two different batches of horse serum. In all cases however, the levels of met-enkephalin-like material (on a per mg protein basis) in clonal cells were markedly lowerthan those found in the mouse hypothalamus at embryonic days 15and. 18 (table 2).

Whether a F7 cell extract or an hypothalamic extract was used, the displacement of specifically bound (1251)-met-enkephalin to antibodies by increasing amounts of endogenous met-enkephalin-like material exhibited the same characteristics as that due to authentic met-enkephalin. As illustrated in fig 3, the displacement curves obtained with the two extracts and the standard are parallel.

Fig. 3

i ’ I , I , I I

Cdl extmct(mll’ 1 ,

Met -enkephalin ( pg/as.ay)

360

Fig 3 : Logit-log inhibition plots of (1251)-met-enkephalin binding to the antiserum II by authentic met-enkephalin (0) or increasing volumes of F7 clonal cell extracts (0) or fetal hypothalamus extracts (x)

Radioimmunoassays were performed with l/15,600 final dilution of antiserum II. In the absence ofunlabelled met-enkephalin, 28-309 of the radioactive ligand were specifically bound to the antiserum (Bo). Non-specific binding (measured in the absence of specific antiserum) corresponded to 5-6% of total radioactivity per sample. The displacement of specifically bound (1251)-met-enkephalin produced by various aliquots of F7 extracts (5-100 ~1 corresponding to 20-450 ng of proteins) or hypothalamic extracts (from 15 day-old mouse embryos-10-100 pl corresponding to 25- 250 pg of proteins i.e. 1/10-l hypothalamus) was measured in duplicates.

Bio-gel P2 chromatography of met-enkephalin-like material in the hypothalamus gave only one peak comigrating with authentic met-enkephalin (fig 4A). The elution profile of met-enkephalin -like material in F7 cell extract was sljghtly more complex : three peaks were apparent (fig 4B). Peaks II and III corresponded to the elution volumes of SO-met-enkephalin and met-enkephalin respectively. Peak I was eluted in the void volume of the gel and could be attributed to immunoreactive canpounds with molecular weights higher than 2,000 daltons.

Fig. 4A

P halin

MOEE HYFoTHALAMl

(ED 15)

Fmctim number

361

Fig. 4B

Fig 4 :

A. The HCl extract of 3 hypothalami from 15 day-old mouse embryos was filtered through a Bio-gel P2 column (54 cm height, 0.9 cm in diameter) with 0.2 N CH3 COOH as the eluent. The collected fractions (0.9 ml each) were lyophilized and assayed for their met-enkephalin-like immunoreactivity. The void volume (19 ml) was determined with cytochrome C.

B. The HCl extract of F7 clonal cells (corresponding to 4.5 mg prot) was filtered through the same column as that described in A. Salts were eluted in fractions 37-43 ; their inhibitory action on the specific binding of (1251)-met-enkephalin to the antiserum (16) mimicks the effect of immunoreactive molecules and likely explains why immunoreactivity was not equal to 0 in these fractions. The elution volumes of met-enkephalin and SO-met-enkephalin were pre-determined with authentic standards.

In both cases (A and B&met-enkephalin-like immunoreactivity is

F7 CELL t i

iXT

2 0.

11 I 14 I I I1 I I 11. 20 Jo 40 50 60 70 80

Fmction number

Bio-gel P2 chromatography of met-enkephalin-like material in fetal mouse hypothalamus (A) and F7 clonal cells (Bf

expressed as pg of met-enkephalin equivalents per fraction.

362

Probable presence of met-enkephalin precursors in F7 clonal cells

Since the met-enkephalin sequence in the peptide presursor(s) is generally sandwich& between two pairs of basic aminoacid residues (see 21 and 22 for the bovine adrenal proenkephalin), trypsin was the appropriate protease for releasing from possible precursors enkephalin molecules (met-enkephalin-lys6, met-enkephalin-arg6, met-enkephalin-arg6-phe7 and gly7-leu8,

met-enkephalin-arg6- see ref 23) which could be recognized by the antiserum

II (table 1).

As shown in table 3, trypsinization of acidic extracts of F7 clonal cells actually induced a marked increase in the amount of immunoreactive material (table 3) migrating like met-enkephalin and SO-met-enkephalin in the Bio-gel P2 column (not shown). In contrast, the same treatment applied to hypothalamic extracts failed to enhance the met-enkephalin-like irmnunoreactivity; surprisingly,3 decrease was even observed (table 3).

In order to verify further the probable presence of met-enkephalin precursors in extracts from F7 cells, the material excluded from the Bio-gel P2 (fig 4B) was treated successively with trypsin and carboxypeptidase B. This combined treatment which releases authentic met-enkephalin molecules from larger precursors (24) produced a marked increase in the amount of met-enkephalin- like materal in fractions containing compounds excluded from the Bio-gel P2 (first experiment : from 23 to 46 pg of met-enkephalin equivalents ; 2nd experiment : equivalents).

from 149 to 344 pq of met-enkephalin

Table 3

Exp: no Met-enkephalin-like material @g/m9 protein)

basal trypsin R

Clonal cells F7 1 50.2 100.7 2.0 2 47.6 105.3 2.2 3 35.9 80.1 2.2 4 85.7 371.7 4.3 m 54.92 10.7 164.5 + 69.3* 2.7 20.5

Fetalhypothalamus ED 15 234.32 30.5 150.9 + 20.0'

66.5% 0.64 + 0.08

ED 18 741.52 64.8 496.0 + 0.67 -0.05

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Table 3 : Effects of trypsin treatment on met-enkephalin-like immunoreactivity in clonal cells F7 and fetal hypothalamus

Trypsin treatment consisted of incubating each extract with 0.25 mg/ml of trypsin for 16 h at 37OC (see “Material and Methods") . Each value is expressed as pg met-enkephalin equivalents per mg protein in the extract before trypsinisation. R is the ratio of the levels of met-enkephalin-like material found after trypsinisation to those ("basal") found in similar samples incubated without trypsin. Each value is the mean of quadrupli- cate determinations (exp. no l-4) or the mean + SEM of at least four separate experiments. f p< 0.05 when compared to the respective basal value.

DISCUSSION

Several observations indicated met-enkephalin :

that F7 clonal cells contain

-Positive immunoreactivity typical of met-enkephalin was detected using two different anti-met-enkephalin sera (I and II) and two different techniques (immunocytochemistry and raditio- assay).

-The immunoreactive material coeluted, at least partly, with authentic met-enkephalin from a Bio-gel P2 column. However the elution profile of met-enkephalin-like material from this column suggested that another peptide (at least) was also present. Among peptides cross-reacting with the met-enkephalin antiserum II, the sulfoxide derivative of met-enkephalin (fig 4B) and met- enkephalin-arg6-phe7 (25) were eluted from similar Bio-gel P2 columns in the same volume as that of the unkncwn peptide (peak II). Other chromatographic techniques including HPLC (26) will be necessary to identify completely the immunoreactive molecule(s) eluted in peak II. However those presentlysuspacted, SO-meizenkephalin and met-enkephalin-arg6-phe7, have been already detected inthe brain of various species and in bovine chromaffin cells (27).

In addition to these small molecules, Bio-gel P2 chromatography revealed immunoreactive compounds with molecular weights at least equal to 2,000. That these compounds might correspond to met-enkephalinprecursors was substantiated by the fact that combined treatment with trypsin and carboxypeptidase B increased their met-enkephalin-like immunoreactivity. Since this treatment is appropriate for releasing the active pentapeptide from larger precursors (24), it could be proposed that met-enkephalin molecules were likely generated from the material excluded from the gel under the present conditions.

364

Not only the combined treatment with trypsin and carboxypeptidase B but also trypsinization alone was sufficient to enhance the met-enkephalin-like immunoreactivity in soluble extracts from F7 clonal cells. This finding is consistent with the probable presence of met-enkephalin precursors since the antiserum presently used for radioimmunoassays (antiserum II) recognized two sequences released by trypsin from such lar e molecules, i.e. phe7 (28).

met-enkephalin-arg6 and met-enkephalin-arg % - Bio-gel P2 chromatography of the met-enkephalin-

like material generated by trypsinization of F7 cell extracts strongly suggested that it corresponded to small peptides with molecular weights close to that of met-enkephalin. Other chromatographic procedures (such asHPLC) will be required to prove that they were in fact met-enkephalin-arg6 and met-enkephalin-arg6-phe7.

Surprisingly, trypsin treatment of soluble extracts from the hypothalamus of mouse embryos did not enhance their met- enkephalin-like immunoreactivity but instead reduced it. Accordingly, met-enkephalin already present in the hypothalamus of 15 day-old fetuses might well not be synthesized but only transported and accumulated inthis region; alternatively, the synthesis of met-enkephalin in the fetal mouse hypothalamus can proceed via a mechanism quite different than that recently dis- covered in the bovine adrenal medulla (21,221. Evidence has been reported already that the brain synthesisof enkephalins does not follow the sequence described for the periphery (29,30). Although these two possibilities would explain why trypsinization did not increase met-enkephalin-like immunoreactivity in hypothalamic extracts, they could not account for the decrease which was in fact observed after such treatment. Whether trypsin in the high range of concentrations presently used could destroy met-enkephalin has yet to be established. However, preliminary findings are in favour of enkephalin cleavage by the protease (Rossier, personal communication).

In addition to met-enkephalin, the clonal cell line F7 contains and synthesizes somatostatin (1). These two peptides were undoubtedly in the same cells since more than 90% of all F7 cells exhibit positive immunoperoxidase reaction colour with specific antisera (fig 2A).

against somatostatin (1) and met-enkephalin Another characteristic of these primitive nerve cells

F7 concerns their capacity to differentiate (at least partially) into neurosecretory neurones related to those synthesizing neurophysin and vasopressin in the hypothalamus (31). Although these observations would indicate that primitive nerve cell lines have pluripotentialities regarding their capacities to synthesize neuroactive molecules, they also suggest that the F7 clone could be used as a simpler model for analysing some peptide-peptide interactions-In this respect, F7 cells would be quite appropriate for verifying the possible negative effect of enkephalins on somatostatin release as hypothesized by Tramu et al. (11)

NEUR- E

who observed the co-occurrence of enkephalins and somatostatin in the same nerve endings of the median eminence. Furthermore, this clone might well be of great potential interest to investigate the functional correlate of co-occurrence of enkephalins and vasopressin (or oxytocin) in hypothalamic neurones projecting to the neurohypophysis (7).

In conclusion, clonal cells related to central (hypothalamic) neurones such as the F7 line would be a valuable preparation to investigate not only the biosynthetic pathway of enkephalins in neurones but also the functional significance of several peptide interactions in the peripheral and central nervous systems.

ACKNOWLEDGEMENTS

The expert technical assistance of Miss F. Artaud and Mr C. Pennarun is gratefully acknowledged.

This research has been supported by grants from INSERM, CNRS and Rhdne Poulenc, S.A.

REFERENCES

1. De Vitry, F., Dubois, M. and Tixier-Vidal, A. (1979). Immu- nological detection of somatostatin in a primitive hypothalamic mouse cell line, precursor of a neurophysin cell lineage. J. Physiol. (Paris) 75 : 11-13.

2. Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A. and Morris, H.R. (1975). Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature (Land.) 258 : 577-579.

3. Hdkfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J.M. and Schultzberg, M. (1980). Peptidergic neurones. Nature (Land.) 284 : 515-521.

4. Glazer, E.J., Steinbusch, H., Verhofstad, A. and Basbaum, A.I. (1981). Serotonin neurons in nucleus raphe dorsalis and paragigantocellularis of the cat contain enkephalin. J. Physiol. (Paris) 77 : 241-245.

5. Viveros, O.H., Diliberto, E.J. Jr., Hazum, E. and Chang, K.J. (1979). Opiate-like materials in the adrenal medulla : evidence for storage and secretion with catecholamines. Mol. Pharmacol. 16 : 1101-1108.

6. Knodel, E.L. and Richelson, E. (1980). Methionine-enkephalin immunoreactivity in fetal rat brain cells in aggregating culture and in mouse neuroblastoma cells.. Brain Res. 197: 565-570.

366

7. Martin, R. and Voigt, K.H. (1981). Enkephalins co-exist with oxytocin and vasopressin in nerve terminals of rat neurohypophysis. Nature (Land.) 289 : 502-504.

8. Erichsen, J.T., Reiner, A. and Karten, H.J. (1982). Co-occurrence of substance P-like and leu-enkephalin-like immunoreacti- vities in neurones and fibres of avian nervous system. Nature (Land.) 295 : 407-410.

9. Lundberg, J.M., Hamberger, B., Schultzberg, M., Hefkfelt, T., Granberg, P.O., EfendiE, S., Terenius, L., Goldstein, M. and Luft, R. (1979). Enkephalin-and somatostatin-like immuno- reactivities in human adrenal medulla and pheochromocytoma. Proc. Natl. Acad. Sci. (USA) 76 : 4079-4083.

10 Tramu, G. and Leonardelli, J. (1979). Immunohistochemical localization of enkephalins in median eminence and adenohypo- physis.Brain Res. 168 : 457-471.

11. Tramu, G., Beauvillain, J.C., Croix, D. and Leonardelli, J. (1981). Comparative immunocytochemical localization of enkephalin and somatostatin in the median eminence, hypothalamus and adjacent areas of the guinea-pig brain. Brain Res. 215 : 235-255.

12. Gilbert, J.A., Knodel, E.L., Stenstrom, S.D. and Richelson, E. (1982). Function and regulation of methionines-enkephalin and its receptors in murine neuroblastoma cells. J. Biol. Chem. 257 : 1274-1281.

13. De Vitry, F., Camier, M., Czernichow, P., Benda, P., Cohen, P. and Tixier-Vidal, A., (1974). Establishment of a clone of mouse hypothalamic neurosecretory cells synthesizing neurophysin and vasopressin. Proc. Natl. Acad. Sci. (USA) 71 : 3575-3579.

14. Gros, C., Pradelles, P., Rouget, C., Bepoldin, O., Dray, F., Fourni+Zaluski, M.C., Rogues, B.P., Pollard, H., Llorens- Cortes, C. and Schwartz, J.C. (1978). Radioimmunoassay of methionine-and leucine-enkephalins in regions of rat brain and comparison with endorphins estimated by a radioreceptor assay. J. Neurochem. 31 : 29-39.

15. Miller,R.J., Chang, K.J., Cooper, R. and Cuatrecasas, P. (1978). Radioimmunoassay and characterization of enkephalins in rat tissue. J. Biol. Chem. 253 : 531-538.

16. Cesselin, F., SoubriC, P., Bourgoin, S., Artaud, F., Reisine, T.D., Michelot, R., Glowinski, J. and Hamon, M. (1981). In vivo release of met-enkephalin in the cat brain. Neuroscience 6 : 301-313.

367

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Tougard, C., Picart, R., Tixier-Vidal, A., Kerdelhue, B. and Jutisz, M. (1974). In situ immunochemical staining of gona- dotropic cells ,in primary cultures of rat anterior pituitary cells with the peroxydase labelled antibody technique. A light and electron microscope study. In : Wisse, E., Daems, W., Molenaar, I., Van Duijn, P. teds) Second International Sympo- sium on Electron Microscopy and Cytochemistry. Amsterdam, North Holland, pp 163-166.

Abraham, G.E. (1969). Solid-phase radioimmunoassay of estra- diol 17 /_3. J. Clin. Endocr. Metab. 29 : 866-870.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 : 265-275.

Snedecor, G.W. and Cochran, W.G. (1967). Statistical methods. Ames. Iowa state College Press (6th edition).

Noda, M., Furutani, Y., Takahashi, H., Toyosato, M., Hirose, T Inayama, S., Nakanishi, S. and Numa, S. (1982). Cloning ani sequence analysis of cDNA for bovine adrenal prepro- enkephalin. Nature (Land.) 295 : 202-206.

Gubler, U., Seeburg, P., Hoffman, B.J., Gage, L.P. and Udenfriend, S. (1982). Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides. Nature (Land.) 295 : 206-208.

Jones, B.N., Shively, J.E., Kilpatrick, D.L., Kojima, K. and Udenfriend, S. (1982). Enkephalin biosynthetic pathway : A 5300-dalton adrenal polypeptide that terminates at its COOH end with the sequence (met)-enkephalin-arg-gly-leu- COOH. Proc. Natl. Acad. Sci. (USA) 79 : 1313-1315.

Lewis, R.V., Stern, A.S., Kimura, S., Rossier,J., St+, S. and Udenfriend, S. (1980). An about 50,000-dalton protein in adrenal medulla : a common precursor of (met)-and (leu)- enkephalin. Science 208 : 1459-1461.

Taquet, H., Javoy-Agid, F., Cesselin, F., Hamon, M., Legrand, J.C. and Agid, Y. (1982). Microtopography of methionine- enkephalin, dopamine and noradrenaline in the ventral mesen- cephalon of human controlard parkinsonian brains. Brain Res. 235 : 303-314.

Cesselin, F., Oliveras, J.L., Bourgoin, S., Sierralta, F., Michelot, R., Besson,J.M. and Hamon, M. (1982). Increased levels of met-enkephalin-like material in the CSF of anaesthe- tized cats after tooth pulp stimulation. Brain Res. 237 : 325-338.

368

27.

28.

29.

30.

31.

Rossier, J., Audigier, Y., Ling, N., Cros, J. and Udenfriend, S. (1980). Met-enkephalin-arg6-phe7, presentin high amounts in brain of rat, cattle and man, is an opioid agonist. Nature (Land.). 288 : 88-90.

Stern, A.S., Lewis, R.V., Kimura, S., Rossier, J., Gerber, L.D., Brink, L., Stein, S. and Udenfriend, S. (1979). Isola- tion of the opioid heptapeptide met-enkephalin. (arg6, phe7) from bovine adrenal medullary granules and striatum. Proc. Natl. Acad. Sci. (USA). 76 : 6680-6683.

Beaumont, A., Fuentes, J.A,, Hughes, J. and Metters, K.M. (1980).0pioid peptide precursors in striatum. FEBS Lett. 122: 135-137.

Boarder, M.R., Lockfeld, A.J. and Barchas, J.D. (1981). Methionine-enkephalin (arg6,phe7) immunoreactivity in bovine chromaffin granules compared with caudate. Brit. J. Pharmacol. 74 : 808 P.

De Vitry, F. (1977). Growth and differentiation of a primitive nervous cell line after in vivo transplantation into syngenic mice. Nature (Land.) 267 : 48-50.

Accepted: 1st June 1982

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Documents

Presence of met-enkephalin in a somatostatin-synthesizing cell line derived from the fetal mouse hypothalamus