Substance P Receptors in Primary Cultures of Cortical Astrocytes from the Mouse (Substance P Analogues/Receptor Binding/Phosphatidylinositol) Y

Home , Bombesin, Carnosine, Kassinin

Proc. Natl. Acad. Sci. USA Vol. 83, pp. 9216-9220, December 1986 Neurobiology Substance P receptors in primary cultures of cortical astrocytes from the mouse (substance P analogues/receptor binding/phosphatidylinositol) Y. TORRENS, J. C. BEAUJOUAN, M. SAFFROY, M. C. DAGUET DE MONTETY, L. BERGSTROM, AND J. GLOWINSKI Chaire de Neuropharmacologie, Institut National de la Sante et de la Recherche MWdicale U.114, College de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 5, France Communicated by Floyd E. Bloom, June 11, 1986

ABSTRACT Binding sites for substance P were labeled on synthesized by S. Lavielle (University Paris VI); thiorphan intact cortical glial cells from newborn mice in primary culture was provided by B. Roques (U.266 Institut National de la using 1251-labeled Bolton-Hunter-labeled substance P. Maxi- Sante et de la Recherche Mddicale, Paris); SP methyl ester mal specific binding (95% of total binding) was reached after was from CRB (Cambridge, England). Other peptides were 2-3 weeks in culture. The binding was saturable, reversible, purchased from Peninsula Laboratories (San Carlos, CA). and temperature dependent. Scatchard and Hill analysis re- 125I-BHSP was obtained by coupling the 125I-BH (Amersham: vealed a single population of noninteracting high-affinity mono-iodo derivative; 2000 Ci/mmol; 1 Ci = 37 GBq) with SP binding sites (Kd, 0.33 nM; B., 14.4 fmol per dish). Com- as described (25). petition studies made with tachykinins and substance P ana- Primary Cultures of Glial Cells. Cells from cerebral cortex logues indicated that the characteristics of the '251-labeled (6 x 105 cells) from 1-day-old Swiss mice (Iffa Credo, St. Bolton-Hunter labeled substance P binding sites on glial cells Germain sur L'Arbresle, France) were dissociated and plated were identical to those on rat brain synaptosomes. 125I-labeled onto culture dishes (16-mm wells) precoated with polyorni- Bolton-Hunter labeled substance P binding sites were visual- thine at 1.5 ,tg/ml (Mr, 40,000; Sigma). The culture medium ized by autoradiography, and differences in the intensity of consisted of a mixture of minimum essential medium (MEM) labeling were seen among astrocytes. Substance P was found to and F12 nutrient (GIBCO) (1:1, vol/vol), supplemented with stimulate phosphatidylinositol turnover; the EC50 value (0.36 33 mM glucose/2 mM glutamine/3 mM NaHCO3/5 mM nM) Was identical to the IC50 value (0.38 nM) determined in Hepes, pH 7.2. The final culture medium, including 10% binding studies. 125I-labeled Bolton-Hunter labeled substance (vol/vol) Nu-serum (Collaborative Research, Waltham, MA), P binding sites were also found on astrocytes derived from was changed every 3 days. Glial cells formed a confluent other brain structures and from the spinal cord of mice. monolayer, devoid of neurones in the 3-week-old cultures used in most of our experiments. Glia from other brain Substance P (SP) receptors have been characterized in the regions were obtained in a similar way. Immunohistochem- central nervous system of adult mammals in binding studies istry using a rabbit antibody against glial fibrillary acidic performed on synaptosomes or membranes using [3H]SP (1, protein (GFAP) (gift from A. Bignami) showed that 95% of 2) or the 125I-labeled Bolton-Hunter (125I-BH) derivative of the cells were GFAP immunoreactive. SP (125I-BHSP) (3-5). The regional localization of these Binding Assays. Routinely, 3- to 4-week-old cultures were binding sites has been shown by microdissection (1, 6) or by incubated for 45 min at 200C with 0.1 nM 125I-BHSP (25,000 autoradiography (7-9), and a correlation has been observed cpm) in the presence or absence of 1 uM SP. The incubating between the distribution of [3H]SP binding sites and the medium (0.2 ml) consisted of a Krebs-Ringer phosphate ability of SP to stimulate phosphatidylinositol turnover (10). buffer (120 mM NaCl/4.8 mM KC1/1.2 mM CaCl2/1.2 mM Using 125I-BHSP, binding sites have also been found on intact MgSO4/15.6 mM NaH2PO4, pH 7) containing bovine serum cells in mixed (glial cells and neurones) primary cultures of albumin (0.4 mg/ml, Calbiochem), 200 ,uM bacitracin (Sig- embryonic mouse (11) or rat (12) brain as well as on neurones ma), 1 ,uM thiorphan, and glucose (6 mg/ml). At the end of in primary culture of embryonic mice (11). Since these the incubation, the supernatant was discarded, and the cells studies did not exclude a localization ofthese binding sites on were washed three times with 0.5 ml of cold Krebs-Ringer astrocytes as well, we investigated the possibility of SP phosphate buffer. Cells were then detached using 0.2% Triton receptors on astrocytes of newborn mice in primary culture. X-100 (0.25 ml) containing bovine serum albumin (1 mg/ml), Indeed, receptors for several neurotransmitters or neuro- and the radioactivity bound to tissues was estimated. Assays hormones have already been found on astrocytes using were performed in quadruplicate. Protein concentrations binding and electrophysiological techniques (13-16), the were determination of adenylate cyclase activity on membranes determined by the method of Lowry et al. (26). (17, 18), or the measurement of cAMP, cGMP (19-21), and In some experiments, the identity of the radioactive ma- inositol phosphates in intact cells (22, 23). In addition, SP was terial recovered in the supernatant at the end of the incuba- shown to amplify the increase in cAMP accumulation evoked tion was checked by reverse-phase HPLC using C18 ,Bond- by norepinephrine in purified astrocytes (24). apak columns and methanol/50 mM ammonium acetate, pH 4 (54:46, vol/vol), as solvent. Light Microscopic Autoradiography. Cortical glial cells MATERIALS AND METHODS were incubated with 1251I-BHSP, washed, fixed for 3 hr under SP and Analogues and Preparation -of 125I-BHSP. SP, paraformaldehyde vapor at room temperature, then covered neurokinin A, neurokinin B, [D-Pro2,D-Trp7'9]SP(1-11) were with Kodak NTB2 emulsion and exposed for 13-27 days at -20°C. Autoradiographs were developed with Kodak Dektol The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: SP, substance P; 125I-BH, '25I-labeled Bolton-Hunter in accordance with 18 U.S.C. §1734 solely to indicate this fact. reagent; GFAP, glial fibrillary acidic protein. 9216 Neurobiology: Torrens et al. Proc. Natl. Acad. Sci. USA 83 (1986) 9217 developer for 3 min at 18'C. After fixation, cells were stained slightly with toluidine blue. Breakdown of Inositol Phospholipids. Three-week-old cortical glial cells were preincubated for 48 hr at 370C (using the culture medium) with 1 ACi ofmyo-[2-3H]inositol per dish (15 Ci/mmol, Amersham). The cultures were washed four times, and Krebs-Ringer phosphate medium (0.5 ml) containing 10 mM LiCl and various concentrations of SP was added for 30 min at 370C. The accumulation of [3H]inositol phosphates was measured by the method of Berridge et al. (27). RESULTS Specific Binding of '2sI-BHSP to Glial Cells. Cortical glial cells from newborn mice (1 postnatal day) were first grown from 1 to 7 weeks in primary culture. Incubations were carried out at 20'C for 45 min with 0.1 nM 125I-BHSP in the absence or presence of 1 ,M SP to determine nonspecific binding. Although low in 1-week-old cultures (20% of the value found in 3-week-old cultures), 1251-BHSP specific binding increased with time and reached a plateau between 2 and 3 weeks. In 3-week-old cultures, the specific binding of 125I-BHSP to glial cells represented about 95% of total binding and 11% of the total amount of 1251-BHSP added into the incubation .-le medium enriched with 200 ,.M bacitracin and 1 AM :t thiorphan. HPLC analysis revealed that 81% of the radioactivity corresponded to 1251-BHSP after a 45-min incubation at 20°C. The amount ofspecific binding of125I-BHSP was nearly identical when the incubation medium (containing 125i- BHSP) from a first 45-min incubation was used a second time with fresh glial cells. C* 1251I-BHSP bound to cortical glial cells was not taken up by a 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, no alteration in the total or specific binding of 125I-BHSP to the cells was observed. Similarly, no modification in 125I-BHSP binding was found when cells were preincubated with 10 AM chloroquine, an inhibitor oflysosomal enzymatic activity that *1 also alters membrane fluidity and hormonal internalization (data not shown). When 3-week-old glial cell cultures from the striatum, thalamus, hypothalamus, cerebellum, mesencephalon, and spinal cord from 1-postnatal-day-old mice were used, a specific binding of 1251I-BHSP similar to that found on cortical FIG 1 Autoradiography of 1251BHSP binding on cortical glial glial cells was observed. cells from the mouse. (A) Astrocytes were grown for 3 weeks in Autoradiography of "2sI-BHSP Binding to Cortical Glial primary culture, and immunohistochemical visualization of GFAP is Cells. Cortical glial cells (3 weeks old) were incubated for 45 shown. (B) The autoradiograph was obtained using 3-week-old cells min at 20°C with a medium containing 0.25 nM 1251-BHSP incubated with 0.25 nM,251-BHSP for 45 mi at 20C. A difference with or without 5 ,M SP. After washing of the cells, in the intensity oflabeling among cells can be noted. (C) Experiments were as indicated above except that 5 A.M SP was added with 125J autoradiographs were prepared. In the absence ofSP, several BHSP. (Bars = 50 pm.) glial cells were labeled highly and uniformly. Although most of the cells were labeled to a certain degree, the intensity of labeling varied from one cell to another (Fig. 1). Since about Using the transformation described by Kitabgi et al. (28), 95% of the cells contained GFAP, it can be assumed that it these data yielded the association (k+1, 5.78 x 108 M-'min'-) was indeed the astrocytes that were labeled with 125I-BHSP. and dissociation (kL1, 1.96 x 10-2 min-') rate constants and No labeled cells were seen when the incubation was made the equilibrium dissociation constant (Kd = klkl= 34 with 5 AM SP. PM). Kinetic Characteristics of 1251-BHSP Specific Binding to When cortical glial cells were incubated with increasing Cortical Glial Cells. The binding of 1251-BHSP to 3-week-old concentrations of 1251-BHSP (from 0.01 to 2.5 nM), nonspe- glial cells reached equilibrium within the 45-min incubation at cific binding (as defined using 1 jxM SP) was a linear function 20°C (Fig. 2). Nonspecific binding was maximal within 15 of the 125I-BHSP concentration. In contrast, the specific min. Specific binding of 125I-BHSP was temperature depen- binding of'1251-BHSP was saturable (Fig. 4, illustration of one dent, being negligible when the incubation was carried out at experiment). Scatchard analysis of data obtained from four 40C (18%). At 200C, 125I-BHSP binding was reversible, since independent experiments made in quadruplicate provided following the initial 45-min incubation dissociation of the average Kd and Bmax, values of 0.33 ± 0.03 nM and 14.4 ±+ 1.5 labeled ligand from its binding sites was observed by substi- fmol per dish (or 170 fmol/mg ofprotein), respectively. When tuting a fresh incubation medium containing 1 AM SP for the the pooled data from all these experiments were analyzed initial incubating medium containing '25I-BHSP (Fig. 3). directly by iterative nonlinear regression analysis, the bind- 9218 Neurobiology: Torrens et al. Proc. Natl. Acad. Sci. USA 83 (1986) (dimethyl-C7) have been shown to have opposing selectivi- 100 3 ties for 125I-BHSP versus 125I-BH-eleodoisin binding sites on .0 rat brain synaptosomes (29), these peptides were also used. CT SP methyl ester was relatively potent in inhibiting 125I-BHSP X binding, while (dimethyl-C7) the metabolically stable ana- logue of SP, showed a very low affinity for 125I-BHSP binding sites (Table 1). E 50- Finally, among all the SP antagonists tested, spantide CO [D-Arg',D-Trp7 9,Leul]SP was found to be the most potent, E but its affinity was only 0.1% that of SP (Table 1). Increased Turnover of Phosphatidylinositol Induced by SPin 0- Cortical Glial Cells. Three-week-old cultures of cortical glial

0 30 60 90 0 15 30 45 cells were incubated for 48 hr with 1 ttCi of myo-[3H]inositol.

Time at min After appropriate washings, SP was added in various con- 200C, centrations for 30 min at 370C in a Krebs medium containing FIG. 2. Association of 125I-BHSP with cortical glial cells. Specific 10mM lithium. SP enhanced the accumulation of [3H]inositol binding of 1251-BHSP was estimated on 3-week-old intact cells. phosphates in a dose-dependent manner, the maximal effect Results are the mean of data obtained in three experiments from (100%o stimulation) being seen at 1 nM. The apparent EC50 quadruplicate samples. Beq concentration of ligand bound at equi- value was estimated to be 0.36 ± 0.07 nM. librium; B, amount of ligand bound at the time considered. k+1, 5.78 x 108 M-1 min-'. DISCUSSION ing parameters were found to be Kd, 0.39 nM and Bma, 15.0 Glial cells possess some voltage-dependent channels (30, 31) fmol per dish, in close agreement to the values estimated by and are able to express receptors for several neurotransmit- Scatchard analysis. These data and the Hill analysis (nH, 1.01 ters or hormones. Among neuropeptides, the presence of ± 0.03) suggested that '251-BHSP bound specifically to a receptors for vasoactive intestinal peptide, opiates, somato- single population of noninteracting binding sites. statin, secretin, calcitonin, or peptides of the corticotropin/ Competition Studies with Tachykinins and SP Analogues. melanotropin family have been suggested (17-21, 32), and Several tachykinins and SP analogues competitively inhibit- this list is not exhaustive. Most ofthese peptide receptors are ed the cortical glial cell high-affinity specific 125I-BHSP coupled positively or negatively to adenylate cyclase. Our binding in a concentration-dependent manner, the Hill coef- study indicates that neonatal cortical glial cells from mice ficient being close to unity for most compounds tested (Table express SP receptors when grown for a few weeks in primary culture. 1). However, no compound was as potent as SP itself (IC50 When 3-week-old cultures of cortical glial cells were = 0.38 + 0.05 nM). Marked differences were seen in the incubated with 125I-BHSP for 45 min at 20°C in the presence potency of the various tachykinins, with the following rank of200 ,uM bacitracin and 1 ,uM thiorphan, minimal peptidasic order of potency: SP > physalaemin > eledoisin > inactivation of 125I-BHSP occurred, and a marked amount of neurokinin A > kassinin > neurokinin B (Fig. 5). A good specific 125I-BHSP binding to the cells was observed. This correlation was found between the length of C-terminal specific binding, which could be detected in younger cultures fragments of SP and their ability to compete with i25I-BHSP but reached its maximal level in confluent 2- to 3-week-old binding, the longer fragments being more potent (Table 1). glial cells was saturable, reversible, and temperature depen- SP-(7-11), which is practically devoid of biological activity, dent. An accumulation of the ligand into the cells through an did not inhibit 125I-BHSP binding. Similar results were found active transport process was excluded since 125I-BHSP bind- with N-terminal fragments of SP (Table 1). ing was not affected by ouabain. Since SP methyl ester and [p-Glu5,MePhe8,Sar91SP(5-11)- Depending on their origin, glial cells may exhibit different properties. Thus, mouse glial cells from the mesencephalon 30 90 150 0 60 120 0 and the striatum but not from the cerebral cortex possess somatostatin receptors coupled negatively to adenylate cyclase (18). This heterogeneity does not seem to occur for 125I-BHSP binding sites that were detected not only on glial cells from the cerebral cortex but on those of all other brain structures examined and ofthe spinal cord as well. 125I-BHSP binding sites were also visualized by autoradiography on E 50 cortical glial cells. Although a quantitative analysis was not E made, most cells seemed to be labeled, but differences in the E intensity of labeling were seen. This could be attributed to variations in apposition of the cells to the emulsion used for .-O~~~~~~~~~~~~~~~~~~- autoradiography, to differences in the morphology, or to the 30 90 150 0 60 120 state of maturation of the cells. The existence of subclasses Time at 200C, min of glial cells characterized by differences in their number of 125I-BHSP binding sites cannot be excluded, since morpho- FIG. 3. Dissociation time course of 125I-BHSP specifically bound logically distinct GFAP-positive cortical astroglia indeed to cortical glial cells. Cells (3 weeks) were incubated with 0.08 nM were shown to differ in whether or not they expressed 25I-BHSP for 45 min at 200C. The incubation medium was then P-adrenergic receptors (15). Since most of the cells were removed and replaced by fresh medium containing 1 ,uM SP. (Left) GFAP positive and since most ofthe cells were labeled, it can The concentration of bound ligand was followed with time. Each be concluded that 125I-BHSP binding sites are located on the point is the mean of data obtained in three experiments from be added that no labeled quadruplicate samples. (Right) Data are represented as ln([B]/[B.]) surface of astrocytes. It should versus time ([B,] concentration of ligand bound before dissociation; neurones were observed on autoradiographs. [B], concentration of bound ligand at time considered) k1, 1.96 x The specific 125I-BHSP binding sites located on cortical 10-2 min-'. astrocytes showed kinetic and pharmacological characteris- Neurobiology: Torrens et al. Proc. Natl. Acad. Sci. USA 83 (1986) 9219

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

'5 0~~~~~~~~~~~~ ? < !~~~~~~~~~~~~~~~~~-. 10 2 CD~~~~~~~~~~~~~~~~~~~~~~~~~~8

c~~~~~~~j~~~~~~~~~~~+3A +4 +5 log free I251-BHSP 1 ~~~2 1251-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-BHSP for 45 min at 20°C. Total (o), nonspecific (A), and specific (o) binding were estimated. Each point represents the mean of quadruplicate obtained in 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 in observed with rat brain synaptosomes, the dissociation primary culture (11) or on rat brain synaptosomes (3, 4). As constant calculated from equilibrium studies (0.33 nM) was higher than that found from kinetic studies (0.034 nM). This Table 1. Relative potencies of various compounds in displacing could result from problems connected with measurements of '251-BHSP on cortical glial cells from the mouse the apparent rates of association and dissociation since a Ki Peptide IC:5, nM n nH (SP) value of0.29 nM was obtained from competition studies. Substance P 0.38 ± 0.05 6 1.00 In any case, 1251-BHSP binding sites had a high affinity, and Physalaemin 1.5 ± 0.1 2 1.00 Scatchard or Hill analyses revealed that 125I-BHSP was Eledoisin 17 ± 1 3 0.88 bound by a single population of noninteracting sites. The Neurokinin A 49 ± 5 3 0.99 Kassinin 53 ± 1 2 0.98 Neurokinin B 100 ± 17 3 0.89 SP-(2-11) 0.66 ± 0.01 2 0.77 SP-(3-11) 2.7 ± 0.3 2 0.86 o 100 - SP-(4-11) 5.8 ± 0.4 2 0.79 SP-(5-11) 12 ± 2 2 0.95 C', SP-(6-11) 18 ± 2 2 0.92 SP-(7-11) >10,000 2 SP-(1-11) free acid 8,800 ± 200 2 1.16 SP-(1-9) free acid >10,000 2 CY SP-(1-9) amide >10,000 2 E SP methyl ester 5.7 ± 1.2 3 0.87 Dimethyl-C7 6,300 ± 1,700 3 0.95 [D-Pro2,D-Trp7'SP(1-11) 1,700 ± 400 3 0.75 0~~0 [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 -11 -10 -9 -8 -7 -6 -5 Cells were incubated for 45 min at 20'C with 0.1 nM '21I-BHSP and log [peptides] increasing concentrations of unlabeled competitive agents. The IC5o values (± SEM) were determined from the Hill plots ofdata obtained FIG. 5. Displacement of 125I-BHSP by tachykinins on cortical in competition studies, results being the mean of n experiments glial cells. Cells (3 weeks) were incubated for 45 min at 20°C with 0.1 (quadruplicate in each case). nH, Hill numbers. Other substances that nM 125I-BHSP and increasing concentrations of the following com- did not compete with 125I-BHSPforbinding sites included thefollowing: pounds: e, SP; *, physalaemin; A, eledoisin; *, neurokinin A; and o, neurotensin, bombesin, bradykinin, somatostatin, metenkephalin, neurokinin B. Data were expressed as the percentage of binding carnosine, and thyrotropin-releasing hormone (all at 10 ,uM), or dopa- obtained in the absence of tachykinins. Each curve is the mean of mine, norepinephrine, serotonin, and GABA (all at 1 mM). three to six experiments with quadruplicate samples. 9220 Neurobiology: Torrens et al. Proc. Natl. Acad. Sci. USA 83 (1986) following conclusions can be drawn from the competition 5. Cascieri, M. A. & Liang, T. (1983) J. Biol. Chem. 258, studies: (i) SP is the most potent tachykinin for displacing 5158-5164. 125I-BHSP binding, the rank order of potencies of the differ- 6. Torrens, Y., Beaujouan, J. C., Viger, A. & Glowinski, J. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 324, 134-139. ent tachykinins being SP > physalaemin > eledoisin > 7. Quirion, R., Shults, C. W., Moody, T. W., Pert, C. B., Chase, neurokinin A > kassinin > neurokinin B. (ii) A good T. N. & O'Donohue, T. L. (1983) Nature (London) 303, relationship exists between the length of SP C-terminal 714-716. fragments and their ability to compete with 251I-BHSP for 8. Shults, C. W., Quirion, R., Chronwall, B., Chase, T. N. & binding, longer fragments are much more potent than shorter O'Donohue, T. L. (1984) Peptides 5, 1097-1128. ones. (iii) The N-terminal fragments of SP are without effect 9. Rothman, R. B., Herkenham, M., Pert, C. B., Liang, T. & on 125I-BHSP binding. (iv) SP antagonists of the undecapep- Cascieri, M. A. (1984) Brain Res. 309, 47-54. tide or octapeptide series are weak inhibitors of 125I-BHSP 10. Mantyh, P. W., Pinnock, R. D., Downes, C. P., Goedert, M. binding. (v) SP methyl ester has a good affinity for the binding & Hunt, S. P. (1984) Nature (London) 309, 795-797. 11. Beaujouan, J. C., Torrens, Y., Herbet, A., Daguet, M. C., sites while dimethyl-C7 does not. Glowinski, J. & Prochiantz, A. (1982) Mol. Pharmacol. 22, SP enhanced the accumulation of labeled inositol phos- 48-55. phates formed from myo-[3H]inositol in 3-week-old cultures 12. Ruegg, V. T. (1983) in Substance P, eds. Skrabanek, P. & of cortical glial cells from the mouse. This effect was Powell, D. (Boole, Dublin), pp. 65-66. concentration dependent, and there was a good agreement 13. Hbsli, E. & Hbsli, L. (1982) Neuroscience 7, 2873-2881. between the EC50 value (0.36 nM) and the IC50 value (0.38 14. Hosli, L., Hbsli, E., Schneider, U. & Wiget, W. (1984) nM) determined in binding studies. Data obtained using rat Neuroscience Lett. 48, 287-291. brain slices (33) and cells from the salivary gland (34) indicate 15. McCarthy, K. D. (1983) J. Pharmacol. Exp. Ther. 226, that SP stimulates phosphatidylinositol turnover. However, 282-290. 16. Hertz, L., Schouboe, I., Hertz, L. & Shousboe, A. (1984) with the latter preparations the ED50 for SP was higher (about Prog. Neuro-Psychopharmacol. Biol. Psychiatry 8, 521-527. 2 orders ofmagnitude) than that found with cortical glial cells. 17. Chneiweiss, H., Glowinski, J. & Prdmont, J. (1985) J. This could suggest a different coupling between the receptor Neurochem. 44, 779-786. protein and the enzyme involved in phosphatidylinositol 18. Chneiweiss, H., Glowinski, J. & Prdmont, J. (1985) J. production in glial cells or an interaction of SP with other Neurochem. 44, 1825-1831. tachykinin receptors that could be present on astrocytes. 19. Van Calker, D., Muller, M. & Hamprecht, B. (1980) Proc. Further experiments undoubtedly will be required to com- Nati. Acad. Sci. USA 77, 6907-6911. pare the pharmacological characteristics of 125I-BHSP bind- 20. Loffler, F., Van Calker, D. & Hamprecht, B. (1982) EMBO J. ing sites and of the receptors mediating the changes in 1, 297-302. 21. Evans, T., McCarthy, K. D. & Harden, T. K. (1984) J. phospholipid metabolism. In any case, results obtained with Neurochem. 43, 131-138. mouse cortical glial cells provide one example of a peptide 22. Pearce, B., Cambray-Deakin, M., Morrow, C., Grimble, J. & receptor involved in the control of phospholipid metabolism Murphy, S. (1985) J. Neurochem. 45, 1534-1540. on glial cells and extend studies that have shown that other 23. Gonzales, R. A., Feldstein, J. B., Crews, F. T. & Raizada, types of receptors (muscarinic, adrenergic) located on M. K. (1985) Brain Res. 345, 350-355. astrocytes were acting through this mechanism (22, 23). 24. Rougon, G., Noble, M. & Mudge, A. W. (1983) Nature (Lon- In conclusion, astrocytes from the neonatal mouse brain don) 305, 715-717. are able to express SP receptors when they are grown in 25. Michelot, R., Gozlan, H., Beaujouan, J. C., Besson, M. J., primary culture. Whether or not these receptors are present Torrens, Y. & Glowinski, J. (1980) Biochem. Biophys. Res. Commun. 95, 491-498. in the adult brain in vivo and what their physiological 26. Lowry, 0., Rosebrough, N., Farr, A. & Randall, R. (1951) J. significance may be in neuronal-glial interactions during Biol. Chem. 193, 265-275. ontogenesis remain to be determined. 27. Berridge, M. J., Downes, C. P. & Hanley, M. R. (1982) Bio- chem. J. 206, 587-595. We thank Dr. H. Chneiweiss who kindly helped us for the 28. Kitabgi, P., Carraway, R., Van Rietschoten, J., Granier, C., preparation of glial cell cultures, as well as Dr. A. Prochiantz, S. Morgat, J. L., Menez, A., Leeman, S. & Freychet, P. (1977) Lavielle, and G. Chassaing for fruitful discussions. This study has been supported by Institut National de la Sante de la Recherche Proc. Nati. Acad. Sci. USA 74, 1846-1850. Mddicale and grants from Centre National de la Recherche 29. Torrens, Y., Beaujouan, J. C. & Glowinski, J. (1985) Neuro- Scientifique (Action Thematique Programmde), Direction des peptides 6, 59-70. Recherches Etudes et Techniques (n085.078), and Rh6ne Poulenc. 30. Gray, P. T. & Ritchie, J. M. (1985) Trends Neurosci. 8, 411-415. 1. Bittiger, H. (1982) Ciba Found. Symp. 91, 195-205. 31. Newman, E. A. (1985) Nature (London) 317, 809-811. 2. Quirion, R. & Pilapil, C. (1984) Neuropeptides 4, 325-329. 32. Lightman, S. L., Ninkovic, M., Hunt, S. P. & Iversen, L. L. 3. Viger, A., Beaujouan, J. C., Torrens, Y. & Glowinski, J. (1983) Nature (London) 305, 235-237. (1983) J. Neurochem. 40, 1030-1039. 33. Watson, S. P. & Downes, C. P. (1983) Eur. J. Pharmacol. 93, 4. Torrens, Y., Lavielle, S., Chassaing, G., Marquet, A., 245-253. Glowinski, J. & Beaujouan, J. C. (1984) Eur. J. Pharmacol. 34. Hanley, M. R., Lee, C. M., Jones, L. M. & Michell, R. H. 102, 381-382. (1980) Mol. Pharmacol. 18, 78-83.