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 Downloaded by guest on September 27, 2021 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 cor- tical 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 radioac- tivity 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.