Striatal proenkephalin turnover and gene transcription are regulated by cyclic AMP and protein kinase c-related pathways

Neuro~cwnce Vol 43, No 1, pp 67 79, 1991 Printed m Great Britain

0306-4522/91 $3 00 + 0 00 Pergamon Press plc ( 1991 IBRO

STRIATAL PROENKEPHALIN TURNOVER AND G E N E T R A N S C R I P T I O N A R E R E G U L A T E D BY CYCLIC AMP AND PROTEIN KINASE C-RELATED PATHWAYS P GIRAUD,* C KOWALSKI,* F. BARTHEL,'~ D BECQUET,* M RENARD,* M GRINO,* F . BOUDOURESQUE* a n d J P LOEFFLER? *Laboratolre de Neuroendocrmologle Exp6nmentale, INSERM U297 affil6e au CRNS, Facult6 de M6decme Nord, Boulevard P Dramard, 13326 Marseflle Cedex 15, France tlnstltut de Physlologle et de Chlmle Blologlque, URA309 du CNRS, Umverslt6 Louis Pasteur, 21 Rue R Descartes, 67084 Strasbourg, France

Abstraet--Preproenkephalin metabohsm, m the rat, was stud~ed m primary strmtal neurons maintained m a chemically defined medmm, Acute treatment (30 mln) with forskohn (10 s M) or phorbol 12 myristate 13 a c e t a t e (10 -7 M) resulted, respectively, m a two- and seven-fold mcrease m methlomne-enkephahn secretmn Chromc treatment with forskohn or phorbol 12 mynstate 13 acetate (24 h) reduced a 100% increase in methlonme-enkephahn content (forskohn) and on the other hand a 50% decrease m methlonmeenkephahn (phorbol 12 myrlstate 13 acetate) Both treatments increased preproenkephahn mRNA levels m a time-dependent manner, this augmentation being observable after 180 mm by Northern blot analysis and in sttu hybridization These data indicate that under chromc stimulation, w~th e~ther forskohn or phorbol 12 mynstate 13 acetate, proenkephahn turnover ~s accelerated However, after stimulation with phorbol 12 mynstate 13 acetate, the more potent methlonme-enkephahn secretagogue, increased peptlde synthes~s ~s not sufficient to replemsh methlonme-enkephahn mtracellular stores Preproenkephahn gene transcription was analysed by introducing the preproenkephahn gene promoter fused to the bacterial acetyl chloramphemcol transferase reporter gene into primary neurons Chronic stimulation (48 h) by forskohn (10 5 M) or phorbol 12 mynstate 13 acetate (10 7 M) of strlatal neurons transfected with th~s fusion gene increased chloramphenlcol acetyltransferase actwlty six-fold and the two effects were ad&tlve These data suggest that the cychc AMP and the protein kmase C pathways &rectly activate preproenkephahn gene transcription

The oplold peptldes m e t h l o n l n e - e n k e p h a h n (MetE N K ) a n d l e u c l n e - e n k e p h a h n as well as their congeners are widely distributed t h r o u g h o u t the central nervous system b u t are particularly c o n c e n t r a t e d m the basal ganglia 222741 U s i n g b o t h light a n d electron microscopic techniques, enkephalin lmmunoreactlvlty was mostly localized in intrinsic medium-sized spiny n e u r o n s of the strlatum. ~7283748 Strlatal e n k e p h a h n erglc neurons project to the external part of the globus palhdus.15 23 M o r e recently, it has u n e q m v o c a l l y been s h o w n by N o r t h e r n b l o t t i n g a n d m s t t u h y b n & z a t l o n techniques that intrinsic strlatal n e u r o n s represented one o f the m a j o r sites for e n k e p h a h n biosynthesis in the central nervous system 7,26,38,44,61 Indeed, h a l f of striatal n e u r o n s express p r e p r o e n k e p h a h n (PPE) m R N A 26 These neurons have been shown to influence nlgrostrlatal d o p a m l n e r g l c p a t h w a y j°'39 a n d to be possibly Involved in physiological a n d / o r physlopathological events of the basal g a n g h a 2,43 __ Ahbrevtattons c-AMP, cychc adenosine monophosphate, CAT, chloramphenlcol acetyltransferase, DMEM, Dulbecco's minimal essential medmm, GFAP, ghal fibrfllary acid protein, HBSS, Hanks' balanced sahne solution, Met-ENK, methlonme-enkephahn, NF, neurofilament, PBS, phosphate-buffered sahne, PKC, protein kmase C, PMA, phorbol 12 mynstate 13 acetate, PPE, preproenkephahn

N u m e r o u s m v w o studies have s h o w n the predomln a n t role of dopamlnerglc transmission on enkephalln t u r n o v e r a n d expression in these structures. 6 ~6,42475262 However, the precise molecular m e c h a n i s m s by which d o p a m i n e modulates oplold biosynthesis remain hypothetlcal A clear u n d e r s t a n d i n g of these m e c h a n i s m s requires a detailed analysis of the second messenger p a t h w a y s involved in this regulation One model a m e n a b l e to such studies is that of strmtal n e u r o n s m p r i m a r y culture Th~s system offers the a d v a n t a g e of investigating b o t h secretory and biosynthetic events in a fully defined e n v i r o n m e n t A n i m p o r t a n t a c c u m u l a t i o n of data supports the i m p h c a t l o n of b o t h cyclic adenosine m o n o p h o s p h a t e (c-AMP) a n d protein klnase C ( P K C ) in the regulatory processes of P P E gene expression ~2 t4 ~8,294549 However, most of these experiments have been conducted in adrenal chromaffin cells or established cell hnes; but these d a t a m i g h t n o t be valid as far as strlatal oploid physiology is concerned This latter question strengthens the choice o f e n k e p h a h n e r g i c enriched p r i m a r y n e u r o n s as a study model Primary stnatal cell cultures are 9 5 0 pure neuronal cells (with less t h a n 4 % astrocytes) u n d e r defined culture conditions used here. Moreover, in this culture system, e n k e p h a h n ~mmunoreactivity is restricted to neuronal cells. These enkephahnerglc neurons acquire 67

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P GIRAUD et al

a satisfactory level o f maturation in terms o f secretory processes and o f peptlde accumulation Indeed, such a model has proven to be extremely useful for a n u m b e r o f pharmacological and biochemical studies o f n e u r o t r a n s m l t t e r receptors and o f their subsequent actlvatlon.S.24.4o.44.5o 54 6o EXPERIMENTAL PROCEDURES Neuronal cell culture

Strlata were dissected out under a stereonucroscope, from 16-day-old Sprague-Dawley rat fetuses obtained from Centre d'Elevage Janxaer (L'Abresles, France) Day of mating was considered as day 0 of gestation Briefly, ussue was resuspended m a small volume 11-2 ml) of a 1' 1 rmxture of Dulbecco's mimmal essential medium (DMEM)-Ham F- 12 nutrient containing 10% fetal calf serum. Dispersion was achieved mechanically by successwe asplratlons through fire-narrowed Pasteur pipettes. Dispersed cells were resuspended m chemically defined medium consisting of a nuxture of DMEM-Ham F-12 (1:1) added with the following ingredients: glucose (25 raM), glutamme (2 mM), msuhn (25#g/ml), transferrin (100 /ag/ml), putrescme (60 #M), progesterone (20 nM), trllodothyronine (1 nM) and selenmm salt (30 riM) This composition is derived from that described by Weiss et al and Bockaert et al for striatal neuron primary cultures'~-6° with some modification from Tlxler-Vldal et al and Fawre-Bauman et al 2°'3~ Cells were plated into 24-well culture plates (Falcon) at a density of 0.5 x 106 cells/cm2 Before plating the cells, culture plates were treated as follows: they were coated with a poly-D-lysine solution (10/~g/ml) in 0.1 M sodium borate buffer (pH 7 4) for 14-16 h at room temperature. After the excess of poly-D-lysme was removed by three successive washes with phosphate-buffered sahne (PBS), pH 7 2, wells were successively coated with a lammin solution m sterde water 110/a g/ml) for 30 nun at room temperature and then w~th 20% fetal calf serum m a nuxture of 1 1 D M E M - F I 2 After this last coating soluUon was removed, the ceils were seeded into the wells (0.5 x 10~ ceUs/cm2). Laminm was prepared and generously gwen by Dr J C Lissitzky (Laboratory of Experimental Cancerology, Marsedle) 3~ Morphological expenments were performed on cells attached on glass coverslips Before seeding of the cells, glass coverslips underwent s~nular treatment as that described for plastic culture plates lmmunocytochemtstry

Immunodetect~on of glml fibnllary acid protein (GFAP) and 200,000 mol wt neurofilament (NF) were performed on ceils fixed w~th cold methanol (for 10mm at -20°C) Specific monoclonal anUbodies (anU-GFAP and anU-NF), purchased from Amersham, were used (accordang to the manufacturer's instructions) at a final dilution of 1-200 and 1-5, respectwely) and incubated for 12 h at 4°C m PBS containmg 1% normal human serum. After three nnses in PBS, primary ant~bo&es were revealed with peroxidase-labelled (Amersham) or fluoresceine-labelled (Jackson) ant~-mouse monoclonal antibodies Preproenkephahn m R N A measurements

Drugs were applied onto the cells on m vttro day 8 Cells were mamtmned in chemically defined medmm for the duratton of the treatment

At the end of each treatment, cells were washed m PBS and lysed m situ w~th 0 3 ml of a solution of 4 M guamdnuum thlocyanate, 25 mM sodmm citrate, pH 7 0, 0 5% sarcosyl. 0 1 M 2-mercaptoethanol following the techmque described by Chomczynskl and Sacchl H w~th a slight mo&ficatlon Sequentially, 0.03 ml of 2 M sodium acetate, pH 4 0. and 0.3 ml of water-saturated phenol were added into the wells The cells were then subrmtted to a brief somcatlon (3 s at low power) and the homogenates corresponding to two wells were pooled m a 1 5-ml rmcrofuge tube conta~mng 0 14 ml of a mixture of chlorofonn-lsoamylalcohol (49 1) After vigorous shaking, the suspension was cooled on ice for 15 mln and centrifuged at 10,000g for 20mm The aqueous phase was nuxed w~th 0.65 ml of isopropanol, placed overmght at - 2 0 ° C and then centrifuged at 10,000 g for 30 mm at 4~C The resulting pellet was washed m 75% cold ethanol, dried under vacuum (3 mm) and subnutted to electrophores~s through formaldehyde-containing agarose gel is ~7 Gels were stained for 5 mm at room temperature with etha&um bronude, destamed for 2 h at 4°C and then photographed under UV hght The s~gnal corresponding to the 28 S rRNA was quantified by dens~tometry of the negatwe of the film (665 polaroid film) Only experiments with a low degree of varmtlon between samples (< 10%) were further explotted RNA was transferred onto nylon membrane tGene Screen) by electroblotting through 0.025 M sodmm phosphate buffer, pH 6 5 (4°C, 14 h, 20 V) m a Blorad "transblot" apparatus PPE mRNA hybridization was performed as previously described, ~s usmg a Pst-I 400-bp rat PPE cDNA fragment (kindly provided by Dr S Sabol), 6~ labelled with [32P]dCTP by random priming (gwmg a final specific radloactwlty of 109 c.p.m//zg DNA) 21 After autoradlography (24-48 h between mtensffymg screens at -80°C), the hybndlzat~on signal was quantified by densltometry (Shlmadzu) Preproenkephahn m R N A m SltU hybrtdlzatwn

Cells were fixed w~th 4% formaldehyde soluuon m PBS for 10 mm at room temperature After three nnses m PBS, cells were acetylated (for 10 nun w~th 0 25% acetic anhydride in 0.1 M tnethanolamme, pH 8 0), then dehydrated by successwe ~mmers~ons(1 mm each unless otherwise indicated) through the following baths 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol, 100% chloroform (5 mm), 100% ethanol, 95% ethanol. Prehybnd~zat~on and PPE mRNA:5 hybridization were performed as prewously described. The same Pstl 400-bp cDNA fragment, labelled w~th [3SS]dCTP by random pnnung was used as a probe Slides were counterstained w~th Tolmdme Blue (0.2%) for 10 s and then dipped into nuclear emulsion (Kodak NTB2) and exposed for three weeks at -80~C Methwnme-enkephahn assay m cellular extracts' and m release medtum

Strmtal neurons were treated between days 8 and 12 of culture For acute treatment (duration < 3 h), chenucatly defined medmm was w~thdrawn and cells were preincubated m release medmm [Hanks' balanced sahne solution (HBSS) containing I mg/ml of bovine serum albumin, 30mM glucose, 1 mM glutamme, 10 -7 M bestatln and 3.10 -6 M th~orphan] for 15 mm Drugs (diluted m release medmm) were then apphed onto the cells When experimental protocol reqmred a longer treatment, drugs were diluted m chenucally defined medmm complemented w~th 10-7 M bestatin and 3 10-6 M th~orphan

F~g I Immunocytolog~cal charactenzaUon of rat strmtal cultures Cells, plated on glass covershps as indicated, were fixed on m wtro day 10 and treated for ~mmunocytocbenustry as indicated m Experimental Procedures (A) 200,000 mol wt NF immunoreact~vtty was revealed w~th fluorescem-labelled ant~-mouse antibody. (B) GFAP-tmmunoreacUve cells were detected using perox~dase-labelled anU-mouse antibody (arrows)

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At the end of the treatment period, incubation medmm was collected, frozen at - 2 0 ° C until assay and the cells were washed with PBS and peptides were extracted with 1 ml of 2 M acetic acid, 0.02 M HCI, and 0 1 M 2-mercaptoethanol After freeze-thawing cycles, the extraction solutlon was transferred into mlcrofuge tubes, boiled for 10 mln, evaporated under vacuum and stored at - 2 0 ' C until assay After solubthzatlon of the dried extract with PBS containing 0 I% bovine serum albumin, Met-ENK was assayed with a sensitive and specific radlo~mmunoassay J6 AntI-Met-ENK antibody was kindly provided by Dr A Cupo Met-ENK secretion was quantified by radlolmmunoassay of 0.1 ml of incubation medmm (without extraction)

Table 1 Effect of forskohn on methiomne-enkephahn secretion from stnatal neurons m primary culture Met-ENK (pg/well per 2 ht Control 6 7 +- () Forskohn 10 6M t27+_16 Forskohn 10-5 M 13 2 + 0 8 PMA 10-7 M 65 +- 7 s PMA 10-7 M + forskohn 10 -6 M 84 5 + 6 5 PMA 10 -7 M + forskohn 10-5 M 88.3 +- 9 2 Experimental conditions were the same as those described in legend of Fig. 2 The mgnificance of the effect of PMA and forskohn was tested by a two-way analysis of vanance PMA action was statistically s]gmficant (Fl8 = 224,

Transfecuon protocol Transfection was performed after six days in culture as prewously reported. 4,33Briefly, the DNA-hpld complex was extemporaneously prepared by mixing 0 5 ml of the plasm~dial DNA solution (pENKAT-12 plasmtd12.2/~g/ml in HBSS) to 0 5 ml of hpopolyamme solution (3 #1 of &octadecylamidoglycylspermlne4 per ml of HBSS) After a 2-h long transcription step, cells were washed and drugs added in chemically defined medmm for 48 h Cells were then lysed and chlorampbenicol acetyltransferase (CAT)actlVlty assayed as previously described 433

This t m m u n o c y t o l o g m a l characterization o f cell types in o u r culture system was i m p o r t a n t to achieve smce type I astrocytes (identified by G F A P labelling) have been s h o w n by others to express a n d regulate P P E 45,54 I m m u n o c y t o c h e m i c a l labelling o f the cells for 200,000 mol. wt N F s u p p o r t the fact that n e u r o n s represent more t h a n 9 0 % of the cells

RESULTS

Immunocytologtcat characterization o f primary culture from fetal rat smatum Striatal p r i m a r y culture, 10 days after seeding, was mainly c o m p o s e d o f neurons, identified o n their capability o f expressing N F lmmunoreactivity, as s h o w n m Fig. IA. Intense labelhng was observed at the neuritic level Cell bodies also displayed N F l m m u n o r e a c t w i t y , b u t w~th a sl]ghter intensity. These n e u r o n s were small- to medium-sized n e u r o n s (soma ranging from 8 to 12/~m m dmmeter). A t the opposite, G F A P - p o s l t i v e cells were scarce ( < 5 % o f the cell population), assembled m groups o f four to six cells (Fig. 1B). These cells, classified as type I astrocytes, have a n extended cytoplasm, as j u d g e d by the raze o f their cytoskeleton. t,,

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P < 0 0001), whereas no statistically slgmficant interaction could be detected between forskohn and PMA (F~ = 1 26) Results are expressed in pg/well of Met-ENK (mean + S E M.. n = 4)

Effects of phorbol 12 myrlstate 13 acetate and forskolin on methtonine-enkephahn secrenon and cellular content m strtatal primary neuronal cell culture A 2-h long exposure of cultured striatal neurons to increasing doses o f p h o r b o l 12 m y n s t a t e 13 acetate ( P M A ) ( f r o m 1 0 - l ° M up to 10 -6 M ) resulted m a dose-related M e t - E N K secretory response. Maximal effect was observed at 10 -7 M with a six-fold increase over basal levels (Fig. 2). Stimulation of M e t - E N K secretion by 1 0 - T M P M A was in the same range o f a m p l i t u d e as t h a t observed with o t h e r secretagogues such as 5 6 m M KCI, 1 0 - 6 M Androctonus austrahs H e c t o r toxin II or 10 5 M N-methyl-D-aspartate (results not shown). U n d e r similar experimental condttions, f o r s k o h n (10 -5 M ) &splayed a shghter effect on M e t - E N K secretion (Table 1), only leading to a two-fold mcrease. This effect was maximal with

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Flg. 2. Met-ENKsecretorydose-responseofcutturedstnatal neurons to PMA. Cells, cultured for 12 days In 24-well plates (0.5 x 106 cells/well) have been treated for 2 h with various doses of PMA. Before the experiment, culture medium was withdrawn and replaced by 0 5 ml of release medium containing enkephalin degrading enzyme mhibitors (see Experimental Procedures) and various doses of effector (laMA in this case). Met-E-NK immunoreactivaty was radiolmmunologlcally assayed Results are expressed in pg/well of Met-ENK (mean 4: S.E M , n = 4)

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Fig 3 Time-course of the action of PMA (10 `7 M) and forskohn (10-SM) on Met-ENK secretion from striatal neurons in primary culture Experimental protocol was identical to that described in the legend of Fig 1, except that cells were treated on day 10 of culture Release medmm was collected from different wells after various incubation periods of time with a constant concentration of drugs. Results are expressed in pg/well of Met-ENK (mean + S E M , n = 4)

Str]atal proenkephahn turnover and gene transcription 1 0 - 6 M forskolln not tested

Lower doses o f this drug were

The secretory response o f enkephahnerglc neurons to P M A was more rapid than w~th forskohn A 30-rmn exposure to 10 7M P M A was suffic]ent to provoke a five-fold increase m M e t - E N K secretion, whereas treatment with 10 -5 M forskolin resulted in a mere 1 5-fold increase o f the secretory response in the same period o f time (Fig 3) Simultaneous apphcat]on o f increasing doses o f P M A with 10 -5 M f o r s k o h n resulted in an additive effect on M e t - E N K secretion (Fig. 4). This additive effect was also observed when 10 6 or 10 5 M

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Table 2 Influence of extracellular calc]um on methlomneenkephahn-mduced secretion from strmtal ne~rons m primary culture Met-ENK (pg/well per 3 h) Control PMA 10 7M PMA l0 7 M + E G T A l m M KCI 56 mM KC1 56 mM + EGTA 1 mM

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Experimental conditions are identical to those described m legend of Ftg 2 (except that cells were treated on m vttro day 8) Results are expressed m pg:well of Met-ENK (mean _+ S E M , n = 4) Significance of the different treatments was tested by analysis of variance followed by SCHEFFE F-test (control vs PMA P < 0 05, PMA + EGTA vs PMA P < 0 05, control vs PMA + EGTA P < 0 0 5 , control vs KC1 P < 0 0 5 , KCI vs KCI+ EGTA P < 0 05, control vs KC1 + EGTA n s )

f o r s k o h n was added to the m e d m m m the presence o f 10 7M P M A (Table 1) The calcium dependency o f P M A effect on peptlde secretion was investigated by applying this drug to cells m calc]um-free m e d m m As s h o w n in Table 2, the increase m M e t - E N K release provoked by phorbol ester was only partmlly d e p e n d e n t upon extracellular calcmm, whereas KC1 stimulation was completely calcmm d e p e n d e n t N o t surprisingly, prelncubat]on o f str]atal neurons with 10 7 M P M A resulted m an important desens]t]zatlon o f M e t - E N K release m response to a further P M A stimulation This p h e n o m e n o n occurred after 15 m m o f P M A premcubat]on (Fig 5) The effect of P M A on enkephahn release corresponded to 40-50% of the ]mtml mtracellular stores o f M e t - E N K Indeed, a 40% decrease of the Met-ENKlmmunoreactwe cell content was observed after a 2h treatment with 10 7M P M A (Fig 6A) whereas no significant changes were not]ced m the case of cells treated with 10 ~ M forskohn (not shown) Interestmgly, mtracellular M e t - E N K xmmunoreactlv]ty remained low after a 24-h exposure of the cells to

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10 4M forskohn resulted In an 80% increase m MetE N K ]mmunoreactwlty (Fig. 6B) Despite the presence of enkephahn degradmg enzyme lnhlb]tors (bestatln and th]orphan), total protection of M e t - E N K lmmunoreactw]ty in the medmm was not achteved using a long mcubaUon period (24 h) It should be noted that the bulk o f the ]mmunoreactwe Met-ENK recovered from either cellular extracts or incubation medium behaved identically to synthetic M e t - E N K on high performance hqmd chromatographic (and not as Met-ENK-related or Met-ENK-extended p e p u d e s ) ( n o t shown) The effects o f second messenger pathway act~vat]on on M e t - E N K secretion and stores p r o m p t e d us to investigate their action on PPE m R N A level variations, A c t t o n o J p h o r b o l 12 m y r t s t a t e 13 acetate a n d / o r s k o h n on p r e p r o e n k e p h a h n m R N A m ~trlatal neuron,s m

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Fig 5 Desenslnzatlon of PMA-stlmulated Met-ENK secrenon m cultured strmtal neurons premcubated with PMA for different periods of time Strlatal neurons were mcubated for various periods of time (as mdlcated m abscissa) with 10 7 M PMA Premcubation medmm was withdrawn and the cells were again exposed to 10-7 M PMA for 15 mm Results are expressed as the ratio of the amount of secreted Met-ENK over the Met-ENK cellular content measured m the cells at the end of the second mcubat]on period (n = 4 )

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o f P P E m R N A was detected as s o o n as 3 h after the drugs were applied o n t o the cells. P P E m R N A levels &splayed a two- a n d three-fold increase after 3 a n d 6 h t r e a t m e n t by b o t h P M A a n d forskolin

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Strlatal proenkephahn turnover and gene transcription

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Fig 8 Absence of effect of depolarizing treatment on PPE mRNA levels in cultured strlatal neurons The hybn&zatlon signal of PPE mRNA of control cells IS shown m 1 Cells treated with 56 mM KC1 for 3 h (con&tlon 2) did not display an increase In PPE mRNA levels By comparison, a 3-h long treatment with 10 5 M forskohn is followed by a clear augmentation of PPE mRNA levels (con&tlon 3) Interestingly, a 3-h-long depolarizing treatment with 56 m M KC1 did not affect PPE m R N A levels (Fig. 8) Despite the purity of our neuronal cell culture (95% pure neurons), it was important to check the nature of the cells in which PPE was expressed and regulated. This was achieved by m s i t u hybridization Twenty per cent of the neuronal cell population is M e t - E N K lmmunoreactive Using m s t t u hybridization we could show an increased density of grams correspondmg to PPE m R N A present on small cells after a 3-h-long treatment with 10 7 M P M A (Fig. 9), thus confirming the results obtained by Northern blotting. Such a PPE m R N A accumulation m neuronal stnatal cells may be the result of R N A stablhzatlon or more probably from increased gene transcription, We did not approach the problem of PPE m R N A stability in our culture model directly In order to examine PPE gene transcription In more detail, we applied a recently developed transfectlon techmque which is particularly well adapted for neuropeptlde gene expression study in such sensitive cells as primary neurons As illustrated In Fig 10, introduction of 1 5 # g of Henk-12 plasmld (consisting of the human PPE promoter fused to the chloramphemcol acetyl transferase gene) into primary strmtal neurons resulted in consistent basal expression of C A T All measurements of C A T activity were carried out 48 h after the cells were transfected. C A T expression was slgnlficantly increased by either P M A or forskolin treatment (10-7 or 10 -5 M, respectively, for 48 h). U n d e r these experimental conditions, the effect of P M A was slightly more intense than that of forskohn However, a strong synergy was observed when cells were treated with both 10 7 M P M A and 10 5 M forskohn This st~mulatory effect appeared to be restricted to a specific subset of genes, since other fusion gene constructs ( H R a s - C A T or Rous Sarcoma wrus long terminal repeat-CAT) were not induced under identical experimental conditions (data not shown), DISCUSSION The tn i, t t r o system we have used, 1 e primary cell culture of fetal rat stnatum, offers a statable model

for the study of neuropeptlde biosynthesis and secretlon, in particular that of enkephahns In a previous set of experiments, we studied the influence of attachment factors on the survival and the maturation of striatal enkephallnerglc neuronal cell in primary culture Although extracellular matrices obtained from bovine corneal endothehal cells proved to be the most favourable substrate in terms of cellular M e t - E N K expression, polylyslne-added lamlnln (which was used in this study) offers a good environment for this type of study Interestingly, the developmental profile of G A B A levels in culture was parallel to that of M e t - E N K 3n The conservation of the ratio of cellular concentrations of G A B A and M e t - E N K between cultured strlatal cells and adult rat strlatum is indicative of a certain degree of normal maturation of the stnatal cell population in our culture conditions, ~° Indeed, others have shown that 5 0 - 6 0 % of GABAerglc rat strlatal neuronal cells m culture coexpressed leucine enkephahn 50 An impressive amount of data has already been accumulated demonstrating the validity of this type of culture system in terms of neurotransmltter receptor activation and coupling of these receptors to functional second messenger pathways, ~:~ ~' ~ and secretory phenomena, s9.6° We have already shown that cultured strlatal cells correctly processed PPE (supported by the identity of M e t - E N K lmmunoreactlwty measured in cell extracts and culture me&a as authentic M e t - E N K after highperformance hquld chromatography charactenzatlonl (results not shown). Maturation of this enkephahnerglc cell population was also demonstrated by their secretory response under depolarizing or Na + channel activation conditions 3o In view of the recent results of other groups, it is important to consider the type of cell (neuronal or ghal cells) expressing enkephahn m primary neuronal culture. Indeed, Vlhjn e t a l ~4 have shown that high levels of PPE m R N A expression could be observed in primary cultured astrocytes As discussed further, the enkephahn gene is regulated by a c-AMP-dependent pathway in astrocytes 4~ Several arguments support the fact that the great majority of enkephahn peptldes and enkephahn m R N A in our culture system comes from neuronal-type cells Firstly, the culture conditions (chemically defined medium and in absence of fetal calf serum) favoured neuronal

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Striatal proenkephahn turnover and gene transcription

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Fig I0 Basal and stimulated expression of transfected pENKAT-12 plasrnld in cultured striatal neurons Cells were transfected as indicated in Experimental Procedures Transfectlon medium was then replaced by chemically defined medium containing with added regulators C, no drugs added, t, 10-7M PMA, f, 10 5 M forskohn, t + f, 10 -7 M PMA and 10 5 M forskohn simultaneously added Cells were lysed after 40 h and CAT activity measured cell development to the detriment of astrocytes J 20,53,57 Indeed, astrocytes identafied as GFAP-posatlve cells, represented less than 4 % of the total cellular populatlon after two weeks m culture, whereas neuronal cells (adentlfied by their positive N F ammunoreactivlty) represented more than 90% of this populataon. MetENK-lmmunoreactwe cells were neuronal as judged, not only by their morphologacal aspect, but also by double ammunocytochermcal labelling (samultaneous immunodetectton of N F and Met-ENK) The neuronal nature of enkephallnerglc cells in strmtal primary cultures was confirmed by Surmeaer e t a l 5o and by Schwartz and Slmanto. 44 It is also clear that M e t - E N K lmmunoreactlvity begins to drop when neuronal cells begin to disappear from the culture and is barely detectable when glial cells overwhelm the culture dish In addition, cell content an terms of M e t - E N K lmmunoreactlvlty was slgnaficantly lower in astrocytlc cultures than in neuronal cell cultures (results not shown), The strikingly elevated levels of PPE m R N A m cultured astrocytes 54 as well as their capacity to express carboxypeptadase E 55 contrast wath the low values of M e t - E N K concentrations. A defect in PPE processing (due to the lack of expression of such endopeptldases which are prohormone-convertlng enzymes or to the lnaccessablllty of PPE to these enzymes) may account for this observation. The exact nature of PPE m R N A - e x p r e s s m g cells is not totally uneqmvocal in our culture system However, It as very hkely that most of PPE m R N A as expressed in neuronal cells, taking into account on the one hand the morphologacal aspect of cells covered with grams

after m s t t u hybri&zatlon for PPE m R N A detection and the hagh percentage of neurons present in culture on the other. Primary culture of neurons removes the complex connections and control mechanisms present m m v t v o or in e x v w o models. The interpretation ofexpenments on neurotransmitter secretion or neurotransmltter receptor actwataon is thus samphfied In these condltaons, the observed stlmulatory effects of both P M A and forskohn on enkephalan secretaon is mdacatlve of the important direct role of these two second messenger pathways in the secretory process of enkephahnergac cell neurons Actwataon of PKC by phorbol ester leads to a dramatic increase in M e t - E N K release It has been demonstrated that synaptlc connections only begin to appear in strmtal culture after the second week m v t t r o . 57 A similar delay 1s observed in hypothalamac neuronal cell culture 3~ Thas effect (as well as the more moderate one observed after forskohn treatment) is very hkely the consequence of the activation of second messenger pathways wathln enkephalmerglc neurons themselves and not that mediated by another neuronal subtype However, indirect mediation of forskohn or P M A effects, vm the secretion of another transmitter into the medium and acting paracrmally. is possable The additive effects of forskohn and P M A on secretory events are suggestive of different sites of action for these two types of drugs Protein klnase C actwlty is known to be present in primary strmtal neuronal cells and has been shown by Weiss e t a l 60 Cellular functions such as mosltol phosphate

Fig 9 In SltU hybridization of PPE mRNA in cultured strlatal neurons Cells were cultured on glass coverslips in chemically defined medium for eight days before they were treated for m s u u hybridization as indicated in Experimental Procedures (A) PPE mRNA hybridization In untreated cells (B) Cells were treated for 3 h with 10 -7 M PMA before fixation Scale bar 20 ~rn

76

P GIR~.UOet

production and KCl-evoked release of GABA are influenced by PKC activity. 6° Here, we show that the secretory machinery m enkephahnergic cells is also strongly influenced by PKC activity, in a dose-related fashion which is similar to that of KCl-sUmulated release of GABA. 6° In stnatal enkephahnergic cells, the st~mulatory effects of PMA on Met-ENK secretion are only parUally dependent upon extracellular calcium. This is not an unprecedented observatlon. Indeed, phorbol ester-stimulated dopamlne release from rat striatal synaptosomes is independent of an increase in cytosohc calcium. 9 In the absence of the measurement of mtracellular calcium levels, ~t is impossible for us to rule out the lmphcatlon of internal calcium m PKC-dependent Met-ENK release from stnatal enkephahnergac neurons (by channel phosphorylation for instance) However, this phenomenon Is controversial and numerous studies have described a synergy between calcium and PKC actwat~on on exocytotlc phenomenon. '165~62 Phorbol esters could enhance the voltage-sensmve influx of calcium 63 In the work presented here, we did not investigate the possible synergy between these two stimulus-secretion coupling pathways It would also be attractwe to study on our culture model the possible existence of PMA-induced long-term MetE N K secretion enhancement as shown by Blttner and Holz m adrenal chromaffin cells5 and to determine the type(s) of PKC isozymes revolved in these phenomena The other second messenger pathway investigated m this study is a c-AMP-dependent pathway The results presented here were obtained by using the adenylate cyclase activator, forskohn Previous studles, partacularly from Bockaert and coworkers, have documented the presence of adenylate cyclase in primary neuronal cells from mouse stnatum and its regulation by various neurotransmltters or related drugs mcludmg dopamme, serotonm,/~-adrenergjc agomsts, vasoactive intestinal polypeptlde and opiold peptldes 8,40,56 In these studies, forskohn was shown to activate c-AMP product10n in a dose-dependent manner (with a maximal effect for a drug concentration of 10 -4 M corresponding to a 30-fold increase m c-AMP formatlon with an EC50 around 10 -5 M). From our results, it is clear that activaUon of c-AMP producUon results m a s~gnlficant increase m enkephahn secretion. Th~s effect ~s, however, quite moderate when compared to that of PMA. Increased Met-ENK secretion occurred for rather low forskolln concentraUon (10 6 M), whereas ~t was reported that only a shght c-AMP accumulation m mouse-cultured stnatal neurons occurred for a s~mllar forskohn concentraUon. Thus, activation of Met-ENK secretion m enkephallnerg~c cells wa c-AMP-dependent mechanism may not be absolutely and hnearly correlated to the amplitude of c-AMP level varmt~on It remains that c-AMPreduced secretion m enkephahnerg~c neurons ts not as exploswe as can be observed under PMA treatment In fact the amphtude of the secretory response

at

of stnatal neurons to 10 -5 M forskohn is comparable to that obtained m hypothalamlc primary neurons for other peptiderglc systems (somatostatm, growth hormone-releasing factor) 3.35 As prewously discussed, maximal PMA and forskohn effects on Met-ENK secretion were additive, suggesting distract sites of action of these second messenger pathway actwators. The mechamsms by which c-AMP accumulaUon reduces Met-ENK secretion m stnatal cells cannot be precisely discussed m view of our results. It is still possible to hypothesize that th~s stimulation may result from ~on-channel activity modification, from interaction with internal calcium-sensitive processes or to direct phosphorylaUon action on protein kmase A phosphorylable targets It should be pointed out that in enkephahnerglc neurons, c-AMP and protein kmase C act positively on enkephalin secreuon m a monodlrectlonal way Indeed, prewous work by Grega e t a l ,~,1 has estabhshed that both phorbol esters and forskohn reduced the same potassium conductance m cultured dorsal root gangha and cerebral hemisphere neurons The importance of the secretory response of enkephalinerglc neurons to PMA should be considered to explain the 40-50% drop m Met-ENK content observed 3 or 24 h after the onset of the stimulation Although PMA leads also to an increased enkephahn biosynthesis (as suggested by the augmentation of PPE m R N A levels)and the induction of pENKAT-12 plasmld transcrtptxon, this enkephahn neosynthesls appears to be insufficient to replemsh the peptlde stores. An identical observation was made after PMA treatment of cultured spinal cord neurons ~9 On the contrary, forskohn (which does not reduce such an ~mportant secretory response of peptlde stores whde leading to a similar increase m PPE m RN A ) led to a s~gnlficant increase m Met-ENK content. It ~s thus possible that PMA-mduced PPE biosynthesis cannot compensate for the profound emptying of peptldlc stores. However, a concomitant action of PMA on the PPE processing cannot be excluded Activation of both the c-AMP- and PKC-dependent pathways have been shown to regulate PPE m R N A levels m bovine adrenal chromaffin cells or m certain transfected cell hnes 12 1,118.29,19With the availability of primary culture of strlatal neurons, it was possible to investigate whether the enkephalin gene would respond similarly to c-AMP or PKC acuvatlon m pure neuronal cells. The results we obtained clearly demonstrate a sigmficant and fast accumulaUon of PPE m R N A in strlatal neurons with both inductors. This rapidity of action was more suggestwe of a transcnpUonat ~mpact of such a treatment than of another mechanism such as m R N A stabdlzauon The direct effect on PPE gene transcription could be demonstrated by using a gene transfer approach. In th~s study, the bacterial plasmld containing the human PPE promoter fused to the CAT reporter gene ~3~,1was introduced into neurons by a techmque previously described. 33Clearly, forskolin and PMA, m an addmve

Striatal proenkephahn turnover and gene transcription

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m a n n e r increased C A T acUvity after 48 h, thus indtcatm g a direct effect o f f o r s k o h n a n d P M A o n P P E gene transcription. Moreover, cycloheximlde treatment, which blocks m R N A t r a n s l a t i o n a n d m a y thus increase m R N A half-life, did n o t change m R N A levels after a 3-h long P M A or f o r s k o h n exposure. In fact, recent w o r k from C o m b et al. s u p p o r t s the ~dea t h a t c - A M P a n d P K C activation b o t h regulate e n k e p h a l i n gene transcription m a positive manner. 12-~4Th~s work perrmtted the ~solation o f a new t r a n s a c t i n g factor n a m e d E N K T F 1 . ~4 In addition, the p o s m v e transacting properties of t h a t protelc complex such as the oncogenes A P l - c - j u n a n d c-fos have also been e s t a b h s h e d for h u m a n e n k e p h a l i n gene regulation 49

e n k e p h a h n - m d u c e d gene regulation xn strlatal neurons clearly demonstrates that the rapid mductxon (less t h a n 4 h) of P P E m R N A after stimulation by forskolin or P M A , is n o t d e p e n d e n t u p o n e a r l y - i m m e d i a t e gene transcription. This unexpected result m i g h t reflect a dxfferent structure in the rat e n k e p h a h n gene 5' p r o m o t e r sequence as c o m p a r e d to the h u m a n gene This problem will n o w be addressed m primary stnatal n e u r o n s during different receptor stimulation In such experiments, i n v o l v e m e n t of early-~mmedlate genes can be analysed by transfectmg cells with expression vectors c o n t a i n i n g sense a n d anti-sense sequences of cloned t r a n s a c t i n g factors [e g c-fos, c-jun a n d c - A M P response element binding protein(s)]

M o r e ewdence for such positwe control comes from experiments using P P E gene a n d oncogenes co-transfected into t e r a t o c a r c i n o m a cell line, 49 these results suggesting t h a t c-fos a n d c-jun m i g h t represent two t r a n s a c t l v a t m g factors l m p h c a t e d m the physiological stimulation of P P E gene transcrlptlon. However, the lack of effect o f cyclohex~mlde on

Acknowledgements--We thank Dr Michael Comb for the gift of pENKAT-12 plasmid We are indebted to Dr Abby Cupo and Dr Steven Sabol for providing us with ant~-MEnk antiserum 2158 and PPE plasmid pRPE2, respectively We thank also Dr J C Lissitzky for the gift of purified laminin. We appreciated the efficacy of Mrs Rosette Querat in typing the manuscript This work was partially supported by grant 6089 from ARC

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Stnatal proenkephahn turnover and gene transcription

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