Proc. Natl. Acad. Sci. USAVol. 83, pp. 9216-9220, December 1986Neurobiology

Substance P receptors in primary cultures of cortical astrocytesfrom the mouse

(substance P analogues/receptor binding/phosphatidylinositol)

Y. TORRENS, J. C. BEAUJOUAN, M. SAFFROY, M. C. DAGUET DE MONTETY, L. BERGSTROM,AND J. GLOWINSKIChaire de Neuropharmacologie, Institut National de la Sante et de la Recherche MWdicale U.114, College de France, 11 place Marcelin Berthelot, 75231 ParisCedex 5, France

Communicated by Floyd E. Bloom, June 11, 1986

ABSTRACT Binding sites for substance P were labeled onintact cortical glial cells from newborn mice in primary cultureusing 1251-labeled Bolton-Hunter-labeled substance P. Maxi-mal specific binding (95% of total binding) was reached after2-3 weeks in culture. The binding was saturable, reversible,and temperature dependent. Scatchard and Hill analysis re-vealed a single population of noninteracting high-affinitybinding sites (Kd, 0.33 nM; B., 14.4 fmol per dish). Com-petition studies made with tachykinins and substance P ana-logues indicated that the characteristics of the '251-labeledBolton-Hunter labeled substance P binding sites on glial cellswere identical to those on rat brain synaptosomes. 125I-labeledBolton-Hunter labeled substance P binding sites were visual-ized by autoradiography, and differences in the intensity oflabeling were seen among astrocytes. Substance P was found tostimulate phosphatidylinositol turnover; the EC50 value (0.36nM) Was identical to the IC50 value (0.38 nM) determined inbinding studies. 125I-labeled Bolton-Hunter labeled substanceP binding sites were also found on astrocytes derived fromother brain structures and from the spinal cord of mice.

Substance P (SP) receptors have been characterized in thecentral nervous system of adult mammals in binding studiesperformed on synaptosomes or membranes using [3H]SP (1,2) or the 125I-labeled Bolton-Hunter (125I-BH) derivative ofSP (125I-BHSP) (3-5). The regional localization of thesebinding sites has been shown by microdissection (1, 6) or byautoradiography (7-9), and a correlation has been observedbetween the distribution of [3H]SP binding sites and theability of SP to stimulate phosphatidylinositol turnover (10).Using 125I-BHSP, binding sites have also been found on intactcells in mixed (glial cells and neurones) primary cultures ofembryonic mouse (11) or rat (12) brain as well as on neuronesin primary culture of embryonic mice (11). Since thesestudies did not exclude a localization ofthese binding sites onastrocytes as well, we investigated the possibility of SPreceptors on astrocytes of newborn mice in primary culture.Indeed, receptors for several neurotransmitters or neuro-hormones have already been found on astrocytes usingbinding and electrophysiological techniques (13-16), thedetermination of adenylate cyclase activity on membranes(17, 18), or the measurement of cAMP, cGMP (19-21), andinositol phosphates in intact cells (22, 23). In addition, SP wasshown to amplify the increase in cAMP accumulation evokedby norepinephrine in purified astrocytes (24).

MATERIALS AND METHODSSP and Analogues and Preparation -of 125I-BHSP. SP,

neurokinin A, neurokinin B, [D-Pro2,D-Trp7'9]SP(1-11) were

synthesized by S. Lavielle (University Paris VI); thiorphanwas provided by B. Roques (U.266 Institut National de laSante et de la Recherche Mddicale, Paris); SP methyl esterwas from CRB (Cambridge, England). Other peptides werepurchased from Peninsula Laboratories (San Carlos, CA).125I-BHSP was obtained by coupling the 125I-BH (Amersham:mono-iodo derivative; 2000 Ci/mmol; 1 Ci = 37 GBq) with SPas described (25).Primary Cultures of Glial Cells. Cells from cerebral cortex

(6 x 105 cells) from 1-day-old Swiss mice (Iffa Credo, St.Germain sur L'Arbresle, France) were dissociated and platedonto culture dishes (16-mm wells) precoated with polyorni-thine at 1.5 ,tg/ml (Mr, 40,000; Sigma). The culture mediumconsisted of a mixture of minimum essential medium (MEM)and F12 nutrient (GIBCO) (1:1, vol/vol), supplemented with33 mM glucose/2 mM glutamine/3 mM NaHCO3/5 mMHepes, pH 7.2. The final culture medium, including 10%(vol/vol) Nu-serum (Collaborative Research, Waltham, MA),was changed every 3 days. Glial cells formed a confluentmonolayer, devoid of neurones in the 3-week-old culturesused in most of our experiments. Glia from other brainregions were obtained in a similar way. Immunohistochem-istry using a rabbit antibody against glial fibrillary acidicprotein (GFAP) (gift from A. Bignami) showed that 95% ofthe cells were GFAP immunoreactive.

Binding Assays. Routinely, 3- to 4-week-old cultures wereincubated for 45 min at 200C with 0.1 nM 125I-BHSP (25,000cpm) in the presence or absence of 1 uM SP. The incubatingmedium (0.2 ml) consisted of a Krebs-Ringer phosphatebuffer (120 mM NaCl/4.8 mM KC1/1.2 mM CaCl2/1.2 mMMgSO4/15.6 mM NaH2PO4, pH 7) containing bovine serumalbumin (0.4 mg/ml, Calbiochem), 200 ,uM bacitracin (Sig-ma), 1 ,uM thiorphan, and glucose (6 mg/ml). At the end ofthe incubation, the supernatant was discarded, and the cellswere washed three times with 0.5 ml of cold Krebs-Ringerphosphate buffer. Cells were then detached using 0.2% TritonX-100 (0.25 ml) containing bovine serum albumin (1 mg/ml),and the radioactivity bound to tissues was estimated. Assayswere performed in quadruplicate. Protein concentrationswere determined by the method of Lowry et al. (26).

In some experiments, the identity of the radioactive ma-terial recovered in the supernatant at the end of the incuba-tion was checked by reverse-phase HPLC using C18 ,Bond-apak columns and methanol/50 mM ammonium acetate, pH4 (54:46, vol/vol), as solvent.

Light Microscopic Autoradiography. Cortical glial cellswere incubated with 1251I-BHSP, washed, fixed for 3 hr underparaformaldehyde vapor at room temperature, then coveredwith Kodak NTB2 emulsion and exposed for 13-27 days at-20°C. Autoradiographs were developed with Kodak Dektol

Abbreviations: SP, substance P; 125I-BH, '25I-labeled Bolton-Hunterreagent; GFAP, glial fibrillary acidic protein.

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Proc. Natl. Acad. Sci. USA 83 (1986) 9217

developer for 3 min at 18'C. After fixation, cells were stainedslightly with toluidine blue.Breakdown of Inositol Phospholipids. Three-week-old cor-

tical glial cells were preincubated for 48 hr at 370C (using theculture medium) with 1 ACi of myo-[2-3H]inositol per dish (15Ci/mmol, Amersham). The cultures were washed four times,and Krebs-Ringer phosphate medium (0.5 ml) containing 10mM LiCl and various concentrations of SP was added for 30min at 370C. The accumulation of [3H]inositol phosphateswas measured by the method of Berridge et al. (27).

RESULTSSpecific Binding of '2sI-BHSP to Glial Cells. Cortical glial

cells from newborn mice (1 postnatal day) were first grownfrom 1 to 7 weeks in primary culture. Incubations werecarried out at 20'C for 45 min with 0.1 nM 125I-BHSP in theabsence or presence of 1 ,M SP to determine nonspecificbinding. Although low in 1-week-old cultures (20% of thevalue found in 3-week-old cultures), 1251-BHSP specificbinding increased with time and reached a plateau between 2and 3 weeks.

In 3-week-old cultures, the specific binding of 125I-BHSP toglial cells represented about 95% of total binding and 11% ofthe total amount of 1251-BHSP added into the incubationmedium enriched with 200 ,.M bacitracin and 1 AMthiorphan. HPLC analysis revealed that 81% of the radioac-tivity corresponded to 1251-BHSP after a 45-min incubation at20°C. The amount of specific binding of 125I-BHSP was nearlyidentical when the incubation medium (containing 125i-BHSP) from a first 45-min incubation was used a second timewith fresh glial cells.

1251I-BHSP bound to cortical glial cells was not taken up bya Na+-, K+-dependent ATPase-coupled process. Indeed,when cells were preincubated for 15 min at 37°C with ouabain(1 mM or 0.1 mM) prior to the addition of the ligand, noalteration in the total or specific binding of 125I-BHSP to thecells was observed. Similarly, no modification in 125I-BHSPbinding was found when cells were preincubated with 10 AMchloroquine, an inhibitor oflysosomal enzymatic activity thatalso alters membrane fluidity and hormonal internalization(data not shown).When 3-week-old glial cell cultures from the striatum,

thalamus, hypothalamus, cerebellum, mesencephalon, andspinal cord from 1-postnatal-day-old mice were used, aspecific binding of 1251I-BHSP similar to that found on corticalglial cells was observed.Autoradiography of "2sI-BHSP Binding to Cortical Glial

Cells. Cortical glial cells (3 weeks old) were incubated for 45min at 20°C with a medium containing 0.25 nM 1251-BHSPwith or without 5 ,M SP. After washing of the cells,autoradiographs were prepared. In the absence of SP, severalglial cells were labeled highly and uniformly. Although mostof the cells were labeled to a certain degree, the intensity oflabeling varied from one cell to another (Fig. 1). Since about95% of the cells contained GFAP, it can be assumed that itwas indeed the astrocytes that were labeled with 125I-BHSP.No labeled cells were seen when the incubation was madewith 5 AM SP.

Kinetic Characteristics of 1251-BHSP Specific Binding toCortical Glial Cells. The binding of 1251-BHSP to 3-week-oldglial cells reached equilibrium within the 45-min incubation at20°C (Fig. 2). Nonspecific binding was maximal within 15min. Specific binding of 125I-BHSP was temperature depen-dent, being negligible when the incubation was carried out at40C (18%). At 200C, 125I-BHSP binding was reversible, sincefollowing the initial 45-min incubation dissociation of thelabeled ligand from its binding sites was observed by substi-tuting a fresh incubation medium containing 1 AM SP for theinitial incubating medium containing '25I-BHSP (Fig. 3).

C*

*1

FIG 1 Autoradiography of 1251BHSP binding on cortical glialcells from the mouse. (A) Astrocytes were grown for 3 weeks inprimary culture, and immunohistochemical visualization of GFAP isshown. (B) The autoradiograph was obtained using 3-week-old cellsincubated with 0.25 nM,251-BHSP for 45 mi at 20C. A differencein the intensity oflabeling among cells can be noted. (C) Experimentswere as indicated above except that 5 A.M SP was added with 125JBHSP. (Bars = 50 pm.)

Using the transformation described by Kitabgi et al. (28),these data yielded the association (k+1, 5.78 x 108M-'min'- )and dissociation (kL1, 1.96 x 10-2 min-') rate constants andthe equilibrium dissociation constant (Kd = klkl= 34PM).When cortical glial cells were incubated with increasing

concentrations of 1251-BHSP (from 0.01 to 2.5 nM), nonspe-cific binding (as defined using 1 jxM SP) was a linear functionof the 125I-BHSP concentration. In contrast, the specificbinding of'1251-BHSP was saturable (Fig. 4, illustration of oneexperiment). Scatchard analysis of data obtained from fourindependent experiments made in quadruplicate providedaverage Kd and Bmax, values of 0.33 ± 0.03 nM and 14.4 ±+ 1.5fmol per dish (or 170 fmol/mg ofprotein), respectively. Whenthe pooled data from all these experiments were analyzeddirectly by iterative nonlinear regression analysis, the bind-

Neurobiology: Torrens et al.

.-le

:t

9218 Neurobiology: Torrens et al.

100 3

.0CTX

E 50-

CO

E

0-

0 30 60 90 0 15 30 45

Time at 200C, min

FIG. 2. Association of 125I-BHSP with cortical glial cells. Specificbinding of 1251-BHSP was estimated on 3-week-old intact cells.Results are the mean of data obtained in three experiments fromquadruplicate samples. Beq concentration of ligand bound at equi-librium; B, amount of ligand bound at the time considered. k+1, 5.78x 108 M-1 min-'.

ing parameters were found to be Kd, 0.39 nM and Bma, 15.0fmol per dish, in close agreement to the values estimated byScatchard analysis. These data and the Hill analysis (nH, 1.01± 0.03) suggested that '251-BHSP bound specifically to a

single population of noninteracting binding sites.Competition Studies with Tachykinins and SP Analogues.

Several tachykinins and SP analogues competitively inhibit-ed the cortical glial cell high-affinity specific 125I-BHSPbinding in a concentration-dependent manner, the Hill coef-ficient being close to unity for most compounds tested (Table1). However, no compound was as potent as SP itself (IC50

= 0.38 + 0.05 nM). Marked differences were seen in thepotency of the various tachykinins, with the following rankorder of potency: SP > physalaemin > eledoisin >neurokinin A > kassinin > neurokinin B (Fig. 5). A goodcorrelation was found between the length of C-terminalfragments of SP and their ability to compete with i25I-BHSPbinding, the longer fragments being more potent (Table 1).SP-(7-11), which is practically devoid of biological activity,did not inhibit 125I-BHSP binding. Similar results were foundwith N-terminal fragments of SP (Table 1).

Since SP methyl ester and [p-Glu5,MePhe8,Sar91SP(5-11)-

30 90 150 0 60 120 0

E 50

E

E

.-O~~~~~~~~~~~~~~~~~~-30 90 150 0 60 120

Time at 200C, min

FIG. 3. Dissociation time course of 125I-BHSP specifically boundto cortical glial cells. Cells (3 weeks) were incubated with 0.08 nM25I-BHSP for 45 min at 200C. The incubation medium was thenremoved and replaced by fresh medium containing 1 ,uM SP. (Left)The concentration of bound ligand was followed with time. Eachpoint is the mean of data obtained in three experiments fromquadruplicate samples. (Right) Data are represented as ln([B]/[B.])versus time ([B,] concentration of ligand bound before dissociation;[B], concentration of bound ligand at time considered) k1, 1.96 x10-2 min-'.

(dimethyl-C7) have been shown to have opposing selectivi-ties for 125I-BHSP versus 125I-BH-eleodoisin binding sites onrat brain synaptosomes (29), these peptides were also used.SP methyl ester was relatively potent in inhibiting 125I-BHSPbinding, while (dimethyl-C7) the metabolically stable ana-logue of SP, showed a very low affinity for 125I-BHSP bindingsites (Table 1).

Finally, among all the SP antagonists tested, spantide[D-Arg',D-Trp7 9,Leul]SP was found to be the most potent,but its affinity was only 0.1% that of SP (Table 1).

Increased Turnover of Phosphatidylinositol Induced by SPinCortical Glial Cells. Three-week-old cultures of cortical glialcells were incubated for 48 hr with 1 ttCi of myo-[3H]inositol.After appropriate washings, SP was added in various con-centrations for 30 min at 370C in a Krebs medium containing10mM lithium. SP enhanced the accumulation of [3H]inositolphosphates in a dose-dependent manner, the maximal effect(100%o stimulation) being seen at 1 nM. The apparent EC50value was estimated to be 0.36 ± 0.07 nM.

DISCUSSION

Glial cells possess some voltage-dependent channels (30, 31)and are able to express receptors for several neurotransmit-ters or hormones. Among neuropeptides, the presence ofreceptors for vasoactive intestinal peptide, opiates, somato-statin, secretin, calcitonin, or peptides of the corticotropin/melanotropin family have been suggested (17-21, 32), andthis list is not exhaustive. Most of these peptide receptors arecoupled positively or negatively to adenylate cyclase. Ourstudy indicates that neonatal cortical glial cells from miceexpress SP receptors when grown for a few weeks in primaryculture.When 3-week-old cultures of cortical glial cells were

incubated with 125I-BHSP for 45 min at 20°C in the presenceof 200 ,uM bacitracin and 1 ,uM thiorphan, minimal peptidasicinactivation of 125I-BHSP occurred, and a marked amount ofspecific 125I-BHSP binding to the cells was observed. Thisspecific binding, which could be detected in younger culturesbut reached its maximal level in confluent 2- to 3-week-oldglial cells was saturable, reversible, and temperature depen-dent. An accumulation of the ligand into the cells through anactive transport process was excluded since 125I-BHSP bind-ing was not affected by ouabain.Depending on their origin, glial cells may exhibit different

properties. Thus, mouse glial cells from the mesencephalonand the striatum but not from the cerebral cortex possesssomatostatin receptors coupled negatively to adenylate cy-clase (18). This heterogeneity does not seem to occur for125I-BHSP binding sites that were detected not only on glialcells from the cerebral cortex but on those of all other brainstructures examined and ofthe spinal cord as well. 125I-BHSPbinding sites were also visualized by autoradiography oncortical glial cells. Although a quantitative analysis was notmade, most cells seemed to be labeled, but differences in theintensity of labeling were seen. This could be attributed tovariations in apposition of the cells to the emulsion used forautoradiography, to differences in the morphology, or to thestate of maturation of the cells. The existence of subclassesof glial cells characterized by differences in their number of125I-BHSP binding sites cannot be excluded, since morpho-logically distinct GFAP-positive cortical astroglia indeedwere shown to differ in whether or not they expressedP-adrenergic receptors (15). Since most of the cells wereGFAP positive and since most ofthe cells were labeled, it canbe concluded that 125I-BHSP binding sites are located on thesurface of astrocytes. It should be added that no labeledneurones were observed on autoradiographs.The specific 125I-BHSP binding sites located on cortical

astrocytes showed kinetic and pharmacological characteris-

Proc. Natl. Acad. Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 9219

.r- 0 ~~~~~~~~~~~~+1 0

? < !~~~~~~~~~~~~~~~~~-.'5 0~~~~~~~~~~~~

10 2CD~~~~~~~~~~~~~~~~~~~~~~~~~~8

c~~~~~~~j A~~~~~~~~~~~+3 +4 +5log free I251-BHSP

1 ~~~21251-BHSP, nM

FIG. 4. Binding of 125I-BHSP to cortical glial cells as a function of '25I-BHSP concentration. Cells (3 weeks) were incubated with 125I-BHSPfor 45 min at 20°C. Total (o), nonspecific (A), and specific (o) binding were estimated. Each point represents the mean of quadruplicate obtainedin one representative experiment. SEM was less than 10o the mean values. (Inset) (Top) Scatchard plot; (Bottom) Hill plot. Kd, 0.275 nM. Bm.,17.46 fmol per dish. nH, 1.02.

tics similar to those found either on embryonic neurones inprimary culture (11) or on rat brain synaptosomes (3, 4). As

Table 1. Relative potencies of various compounds in displacing'251-BHSP on cortical glial cells from the mouse

Peptide IC:5, nM n nH

Substance P 0.38 ± 0.05 6 1.00Physalaemin 1.5 ± 0.1 2 1.00Eledoisin 17 ± 1 3 0.88Neurokinin A 49 ± 5 3 0.99Kassinin 53 ± 1 2 0.98Neurokinin B 100 ± 17 3 0.89SP-(2-11) 0.66 ± 0.01 2 0.77SP-(3-11) 2.7 ± 0.3 2 0.86SP-(4-11) 5.8 ± 0.4 2 0.79SP-(5-11) 12 ± 2 2 0.95SP-(6-11) 18 ± 2 2 0.92SP-(7-11) >10,000 2SP-(1-11) free acid 8,800 ± 200 2 1.16SP-(1-9) free acid >10,000 2SP-(1-9) amide >10,000 2SP methyl ester 5.7 ± 1.2 3 0.87Dimethyl-C7 6,300 ± 1,700 3 0.95[D-Pro2,D-Trp7'SP(1-11) 1,700 ± 400 3 0.75[D-Arg',D-Trp7'9,Leu11]SP(1-11) 330 ± 90 3 0.94[D-Pro4,D-Trp7'ISP(4-11) 2,500 ± 800 2 0.77[D-Pro4,D-Trp7'9"01SP(4-11) 7,300 ± 700 2 0.73

Cells were incubated for 45 min at 20'C with 0.1 nM '21I-BHSP andincreasing concentrations of unlabeled competitive agents. The IC5ovalues (± SEM) were determined from the Hill plots of data obtainedin competition studies, results being the mean of n experiments(quadruplicate in each case). nH, Hill numbers. Other substances thatdid not compete with 125I-BHSPforbinding sites included the following:neurotensin, bombesin, bradykinin, somatostatin, metenkephalin,carnosine, and thyrotropin-releasing hormone (all at 10 ,uM), or dopa-mine, norepinephrine, serotonin, and GABA (all at 1 mM).

observed with rat brain synaptosomes, the dissociationconstant calculated from equilibrium studies (0.33 nM) washigher than that found from kinetic studies (0.034 nM). Thiscould result from problems connected with measurements ofthe apparent rates of association and dissociation since a Ki(SP) value of0.29 nM was obtained from competition studies.In any case, 1251-BHSP binding sites had a high affinity, andScatchard or Hill analyses revealed that 125I-BHSP wasbound by a single population of noninteracting sites. The

o 100 -

C',

CY

E

0~~0

-11 -10 -9 -8 -7 -6 -5log [peptides]

FIG. 5. Displacement of 125I-BHSP by tachykinins on corticalglial cells. Cells (3 weeks) were incubated for 45 min at 20°C with 0.1nM 125I-BHSP and increasing concentrations of the following com-pounds: e, SP; *, physalaemin; A, eledoisin; *, neurokinin A; and o,neurokinin B. Data were expressed as the percentage of bindingobtained in the absence of tachykinins. Each curve is the mean ofthree to six experiments with quadruplicate samples.

Neurobiology: Torrens et al.

9220 Neurobiology: Torrens et al.

following conclusions can be drawn from the competitionstudies: (i) SP is the most potent tachykinin for displacing125I-BHSP binding, the rank order of potencies of the differ-ent tachykinins being SP > physalaemin > eledoisin >neurokinin A > kassinin > neurokinin B. (ii) A goodrelationship exists between the length of SP C-terminalfragments and their ability to compete with 251I-BHSP forbinding, longer fragments are much more potent than shorterones. (iii) The N-terminal fragments of SP are without effecton 125I-BHSP binding. (iv) SP antagonists of the undecapep-tide or octapeptide series are weak inhibitors of 125I-BHSPbinding. (v) SP methyl ester has a good affinity for the bindingsites while dimethyl-C7 does not.SP enhanced the accumulation of labeled inositol phos-

phates formed from myo-[3H]inositol in 3-week-old culturesof cortical glial cells from the mouse. This effect wasconcentration dependent, and there was a good agreementbetween the EC50 value (0.36 nM) and the IC50 value (0.38nM) determined in binding studies. Data obtained using ratbrain slices (33) and cells from the salivary gland (34) indicatethat SP stimulates phosphatidylinositol turnover. However,with the latter preparations the ED50 for SP was higher (about2 orders ofmagnitude) than that found with cortical glial cells.This could suggest a different coupling between the receptorprotein and the enzyme involved in phosphatidylinositolproduction in glial cells or an interaction of SP with othertachykinin receptors that could be present on astrocytes.Further experiments undoubtedly will be required to com-pare the pharmacological characteristics of 125I-BHSP bind-ing sites and of the receptors mediating the changes inphospholipid metabolism. In any case, results obtained withmouse cortical glial cells provide one example of a peptidereceptor involved in the control of phospholipid metabolismon glial cells and extend studies that have shown that othertypes of receptors (muscarinic, adrenergic) located onastrocytes were acting through this mechanism (22, 23).

In conclusion, astrocytes from the neonatal mouse brainare able to express SP receptors when they are grown inprimary culture. Whether or not these receptors are presentin the adult brain in vivo and what their physiologicalsignificance may be in neuronal-glial interactions duringontogenesis remain to be determined.

We thank Dr. H. Chneiweiss who kindly helped us for thepreparation of glial cell cultures, as well as Dr. A. Prochiantz, S.Lavielle, and G. Chassaing for fruitful discussions. This study hasbeen supported by Institut National de la Sante de la RechercheMddicale and grants from Centre National de la RechercheScientifique (Action Thematique Programmde), Direction desRecherches Etudes et Techniques (n085.078), and Rh6ne Poulenc.

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Proc. Natl. Acad. Sci. USA 83 (1986)