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• W2046906382 abstract "In the present study, 3-day treatment of nondifferentiated NG108-15 cells with 100 nM angiotensin II (Ang II) induces morphological differentiation of neuronal cells characterized by the outgrowth of neurites. These morphological changes are correlated with an increase in the level of polymerized tubulin and in the level of the microtubule-associated protein, MAP2c. Mediation by the AT2 receptor may be inferred since: (a) these cells contain only AT2 receptors; (b) the effects are mimicked by CGP 42112 (an AT2 receptor agonist); (c) they are not suppressed by the addition of DUP 753 (an AT1 receptor antagonist); and (d) are abolished by co-incubation with PD 123319 (an AT2 receptor antagonist). Application of Ang II in dibutyryl cAMP-differentiated cells (which contain both types of receptors) induces neurite retraction, an effect mediated by the AT1 receptor. These results indicate that the AT2 receptor of Ang II induces neuronal differentiation, which is initiated through an increase in the levels of MAP2c associated with tubulin. Moreover, our results demonstrate that the AT1 receptor inhibit the process of differentiation induced by dibutyryl cAMP, whereas the AT2 receptors potentiate this effect, illustrating negative cross-talk interaction between the two types of Ang II receptors. In the present study, 3-day treatment of nondifferentiated NG108-15 cells with 100 nM angiotensin II (Ang II) induces morphological differentiation of neuronal cells characterized by the outgrowth of neurites. These morphological changes are correlated with an increase in the level of polymerized tubulin and in the level of the microtubule-associated protein, MAP2c. Mediation by the AT2 receptor may be inferred since: (a) these cells contain only AT2 receptors; (b) the effects are mimicked by CGP 42112 (an AT2 receptor agonist); (c) they are not suppressed by the addition of DUP 753 (an AT1 receptor antagonist); and (d) are abolished by co-incubation with PD 123319 (an AT2 receptor antagonist). Application of Ang II in dibutyryl cAMP-differentiated cells (which contain both types of receptors) induces neurite retraction, an effect mediated by the AT1 receptor. These results indicate that the AT2 receptor of Ang II induces neuronal differentiation, which is initiated through an increase in the levels of MAP2c associated with tubulin. Moreover, our results demonstrate that the AT1 receptor inhibit the process of differentiation induced by dibutyryl cAMP, whereas the AT2 receptors potentiate this effect, illustrating negative cross-talk interaction between the two types of Ang II receptors. Pharmacological studies have clearly identified two classes of angiotensin II (Ang II) 1The abbreviations used are: Ang IIangiotensin IIMAPmicrotubule-associated proteindbcAMPdibutyryl cAMPFBSfetal bovine serumHBSHanks' buffered salinePIPES1,4-piperazinediethanesulfonic acidTBSTris-buffered salineSarsarcosineDMEMDulbecco's modified Eagle's medium. receptors. The AT1 receptor is closely associated with cardiovascular regulation, fluid volume homeostasis, and cellular growth (1Bottari S.P. de Gasparo M. Steckelings U.M. Levens N.R. Front. Neuroendocrinol. 1993; 14: 123-171Google Scholar, 2Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Say J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251Google Scholar). Activation of the AT1 receptor is linked to phospholipase C activation and Ca2+ influx, effects which are mediated by G proteins (1Bottari S.P. de Gasparo M. Steckelings U.M. Levens N.R. Front. Neuroendocrinol. 1993; 14: 123-171Google Scholar, 2Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Say J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251Google Scholar). The AT2 receptors have been identified in many fetal tissues, including brain (3Gehlert D.R. Gackenheimer S.L. Schober D.A. Neuroscience. 1991; 44: 501-514Google Scholar, 4Obermüller N. Unger T. Culman J. Gohlke P. De Gasparo M. Bottari S.P. Neurosci. Lett. 1991; 132: 11-15Google Scholar, 5Millan M.A. Jacobowitz D.M. Aguilera G. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11440-11444Google Scholar, 6Tsusumi K. Strümberg C. Viswanathan M. Saavedra J.M. Endocrinology. 1991; 129: 1075-1082Google Scholar, 7Tsutsumi K. Saavedra J.M. Mol. Pharmacol. 1992; 41: 290-297Google Scholar, 8Barnes J.M. Lucinda J.S. Barber P.C. Barnes N.M. Eur. J. Pharmacol. 1993; 230: 251-258Google Scholar, 9Heemskerk F.M.J. Zorad S. Seltzer A. Saavedra J.M. Neuroreport. 1993; 4: 103-105Google Scholar), and in cell lines of neuronal origin (10Speth R.C. Mei L. Yamamura H.I. Pept. Res. 1989; 2: 232-239Google Scholar, 11Webb M.L. Liu E.C.-K. Cohen R.B. Hedberg A. Bogosian E.A. Monshizadegan C.M. Serafino R. Moreland S. Murphy T.J. Dickinson K.E.J. Peptides. 1992; 13: 499-508Google Scholar, 12Leung K.H. Roscoe W.A. Smith R.D. Timmermans P.B.M.W.M. Chiu A.T. Eur. J. Pharmacol. 1991; 227: 63-70Google Scholar). This receptor has been cloned (13Mukoyama M. Nakajima M. Horiuchi M. Sasamura H. Pratt R.E. Dzau V.J. J. Biol. Chem. 1993; 268: 24539-24542Google Scholar, 14Kambayashi Y. Bardhan S. Takahashi K. Tsuzuki S. Inui H. Hamakubo T. Inagami T. J. Biol. Chem. 1993; 268: 24543-24546Google Scholar), but a definitive physiological function has yet to be assigned. We have reported previously that Ang II decreases the T-type calcium current in nondifferentiated NG108-15 cells expressing only the angiotensin AT2 receptor type (15Buisson B. Bottari S.P. de Gasparo M. Gallo-Payet N. Payet M.-D. FEBS Lett. 1992; 309: 161-164Google Scholar, 16Buisson B. Laflamme L. Bottari S.P. de Gasparo M. Gallo-Payet N. Payet M.D. J. Biol. Chem. 1995; 270: 1670-1674Google Scholar). Kang et al. (17Kang J. Posner P. Sumners C. Am. J. Physiol. 1994; 267: C1389-C1397Google Scholar) also reported that activation of the AT2 receptor increased a potassium channel activity. Moreover, Xiong and Marshall (18Xiong H. Marshall K.C. Neuroscience. 1994; 62: 163-175Google Scholar) reported that Ang II, via the AT2 receptor, could inhibit membrane depolarization and action potential elicited by N-methyl-D-aspartate receptors in the locus coeruleus, a brain area containing only Ang II receptors of the AT2 subtype. Considering the abundance of T-type Ca2+ channels in neurons from fetal brain (19Spitzer N.C. J. Neurobiol. 1991; 22: 659-673Google Scholar), the crucial role of Ca2+ in neuronal differentiation (20Kater S.B. Mills L.R. J. Neurosci. 1991; 11: 891-899Google Scholar) and the abundance of AT2 receptors during this developmental period, it could be postulated that Ang II, via the AT2 receptor could affect some aspects of neuronal differentiation. angiotensin II microtubule-associated protein dibutyryl cAMP fetal bovine serum Hanks' buffered saline 1,4-piperazinediethanesulfonic acid Tris-buffered saline sarcosine Dulbecco's modified Eagle's medium. Neuronal differentiation is characterized by neurite extension that involves several biochemical steps directed toward promotion of the assembly of tubulin monomers into microtubules necessary to support the growing neurites. Several molecules play crucial roles in neurite outgrowth. Among these are the microtubule-associated proteins (MAPs) (21Tucker R.P. Brain Res. Rev. 1990; 15: 101-120Google Scholar), which include both high molecular weight proteins, termed MAP1 to MAP5, and low molecular weight proteins, including tau (22Matus A. Annu. Rev. Neurosci. 1988; 11: 29-44Google Scholar). These proteins promote tubulin polymerization as well as stabilize microtubules and occur as embryonic and adult isoforms whose differential expression during brain development correlates with the maturation of neuronal circuitry. For example, MAP2 and tau bind to distinct populations of microtubules in adult neurons: MAP2 to somatodendritic microtubules and tau to axonal microtubules (23Matus A. Bernhardt R. Hugh-Jones T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3010-3014Google Scholar, 24Binder L.I. Frankfuter A. Rebhun L.I. J. Cell Biol. 1985; 101: 1371-1378Google Scholar). Expression of specific brain MAPs is critical for regulating neurite outgrowth and differentiation. Several lines of evidence have demonstrated a strong correlation between the pattern of expression of neuronal MAPs and the morphological differentiation of neurons (22Matus A. Annu. Rev. Neurosci. 1988; 11: 29-44Google Scholar). In their nondifferentiated state, the hybrid cells NG108-15 (neuroblastoma × glioma) are rounded and actively dividing. Chronic exposure of NG108-15 cells to dibutyryl cAMP (dbcAMP) induces a process of differentiation that includes elaboration of neurites, development of electrical excitability, formation of functional synapses, alteration of ligand-gated channel properties, and a reduced rate of cell division. Thus, differentiated NG108-15 cells exhibit a neuronal phenotype, whereas glial properties appear to be suppressed (25Hamprecht B. Int. Rev. Cytol. 1977; 49: 99-170Google Scholar, 26Hamprecht B. Glaser T. Reiser G. Bayer E. Propst F. Methods Enzymol. 1985; 109: 316-341Google Scholar). Moreover, when nondifferentiated, NG108-15 cells express exclusively the AT2 receptor subtype and mainly the T-type Ca2+ channel (15Buisson B. Bottari S.P. de Gasparo M. Gallo-Payet N. Payet M.-D. FEBS Lett. 1992; 309: 161-164Google Scholar). These cells also express tau and MAP2 and are thus useful in examining factors initiating neuronal differentiation (27Beaman-Hall C.M. Vallano M.L. J. Neurobiol. 1993; 24: 1500-1516Google Scholar). We have taken advantage of these properties to investigate the role of Ang II on differentiation and neurite outgrowth, with emphasis on cytoskeletal proteins. We found that Ang II induces outgrowth of neurites in nondifferentiated cells. These morphological changes are correlated with an increase in polymerized tubulin and an increase in the level of microtubule-associated MAP2c. In contrast, during the process of differentiation with dbcAMP, where both AT1 and AT2 receptors are present, application of Ang II induces neurites involution. These results are the first to assign a physiological role for the AT2 receptor in neuronal differentiation, which is to induce neurite outgrowth, by acting on tubulin polymerization and on the level of microtubule-associated MAP2c. Moreover, our results demonstrate that activation of the AT1 receptor subtype inhibits the process of differentiation induced by dbcAMP and inhibits the effect elicited by the AT2 receptor. NG108-15 cells (provided by Drs. M. Emerit and M. Hamon; INSERM, U. 238, Paris, France) were cultured (passage 4 to 17) in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc., Burlington, Canada) with 10% fetal bovine serum (FBS, Life Technologies, Inc.), HAT supplement (hypoxanthine, aminopterin, thymidine) (Life Technologies, Inc.), and 50 mg/liter gentamycin at 37°C in 80-cm2 Nunclon Delta flasks in a humidified atmosphere of 93% air and 7% CO2, as suggested by Hamprecht et al. (26Hamprecht B. Glaser T. Reiser G. Bayer E. Propst F. Methods Enzymol. 1985; 109: 316-341Google Scholar). The medium was replaced every 2 days. Subcultures were performed at subconfluence. Under these conditions, cells express only the AT2 receptor subtype of Ang II (Fig. 1A). For differentiation, the cells were cultured for 24 h in the normal culture medium and thereafter replaced by DMEM low glucose (1 mg/liter glucose) containing 1% fetal calf serum (Inotech), HAT supplement, 50 mg/liter gentamycin, and 1 mM dbcAMP (Boehringer Mannheim, Montreal, Canada). The cells were allowed to differentiate for 24 h prior to Ang II or Ang II/analogs treatment. Cells were cultured for 3 subsequent days under these conditions, with the differentiation medium being changed every day. For all experiments, cells were plated at the same initial density. Experiments and cytoskeleton extractions were performed on the 4th day. The analog [Sar1,Ile8]Ang II was iodinated by the Iodogen method and separated on a 25-cm C-18 µ-Bondapak column (Waters, Milford, MA) with a linear gradient of 20-60% acetonitrile in a buffer of 7% isopropanol, 0.25 M ammonium acetate, pH 5.0, at a flow rate of 1 ml/min. Carrier-free monoiodinated product was obtained as a single homogenous peak at 20 min. The specific activity was approximately 1000 Ci/mmol. Binding assays were performed on cultured cells as described previously (28Guillon G. Gallo-Payet N. Balestre M.-N. Lombard C. Biochem. J. 1986; 253: 765-775Google Scholar). NG108-15 cells (1.0-1.5 × 106 cells/Petri dish) were washed with 2 ml of Hanks' buffered saline (HBS: NaCl, 130 mM; KCl, 3.5 mM; CaCl2, 1.8 mM; MgCl2, 0.5 mM; NaHCO3, 2.5 mM; HEPES, 5 mM, supplemented with 1 g/liter glucose and 0.5% BSA) and incubated for 15 min at 22°C in the same medium. The hormone binding reaction was initiated by quick aspiration of the HBS medium and addition to each Petri dish of 0.8 ml of HBS containing the labeled peptide alone or with analogs as described above. Incubations were performed in triplicates for 45 min at room temperature (22°C). After incubation, cells were rapidly detached by scraping them with a rubber policeman. Incubations media were filtered through Whatman GF/C filters, rinsed three times, and counted in a Beckman γ counter. Displacement curves illustrated in Fig. 1 show that, in nondifferentiated cells, DUP 753 did not displace the iodinated Ang II analog from the Ang II receptor (Fig. 1A), although, in differentiated cells, labeling was displaced by DUP 753 (Fig. 1B), indicating the presence of AT1 receptors. These results show that nondifferentiated cells contain only AT2 receptors, while cells that have initiated differentiation express both subtypes of the Ang II receptor. Three days of Ang II treatment induced the expression of AT1 receptors, which remained in low proportions however (20% displacement) compared with the AT2 receptors (Fig. 1C), while decreasing the number of AT1 receptors in dbcAMP-differentiated cells (20% displacement compared with 50% in control dbcAMP-treated cells) (Fig. 1D). Preparations enriched in microtubules were obtained from cells grown in 100-mm Petri dishes as described by Solomon (29Solomon F. Methods Enzymol. 1986; 134: 139-147Google Scholar) with some modifications. The cells were pretreated with 1 µM Taxol (Sigma) for 2 h before extraction of microtubules. At this concentration Taxol stabilizes microtubules without promoting polymerization. The culture medium was then aspirated and replaced by PM2G buffer (PIPES, 0.1 M; glycerol, 2 M; MgCl2, 5 mM; EGTA, 2 mM, aprotinin, 40 TIU/ml; phenylmethylsulfonyl fluoride, 2 mM; benzamidine, 1 mM, pH 6.9) containing Taxol (1 µM). Cells were scraped from the substratum with a rubber policeman and transferred to a 15-ml conical tube and centrifuged at 1000 × g for 5 min at 37°C. The cell pellet was then extracted with PM2G buffer containing 1% Nonidet P-40 and 1 µM Taxol. After a 15-min incubation at 37°C the suspension was centrifuged at 1000 × g for 5 min at 37°C. The pellet containing the microtubules and associated proteins was then solubilized in Tris buffer 125 mM, pH 6.8, containing 4% sodium dodecyl sulfate (SDS) (w/v), 20% glycerol (v/v), and 10%β-mercaptoethanol (v/v) and heated to 100°C for 5 min. After centrifugation at 10,000 × g for 5 min, the supernatant was stored at -20°C until Western blot analysis. For total cell extracts, cells grown in 100-mm Petri dishes were washed twice with HBS buffer and solubilized as described above. β-Tubulin was detected with monoclonal antibody purchased from Amersham (Oakville, Ontario, Canada); monoclonal antibody that recognizes all MAP2 forms were purchased from Sigma and the monoclonal tau antibody 5E2 was kindly provided by Dr. Ken Kosik (Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA). Samples from equivalent number of cells were compared in each experiment. Samples were separated on 4-15% (w/v) SDS-polyacrylamide gels. Proteins were transferred electrophoretically to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, CA). Membranes were blocked with 1% gelatin, 0.05% Tween 20 in TBS buffer, pH 7.5. After three washes with TBS/Tween 20 (0.05%), membranes were incubated either with anti-tubulin (1:250), anti-MAP2 (1:500), or anti-tau (1:750) for 2 h at room temperature, followed by four washes with TBS/Tween 20. Detection was accomplished using horseradish peroxidase-conjugated anti-mouse antibody (Amersham, Oakville, Ontario, Canada) and enhanced chemiluminescence (ECL) detection system (Amersham). The immunoreactive bands were scanned by laser densitometry and expressed in arbitrary units. For immunofluorescence, cells were plated on plastic coverslips (Starsted, St. Laurent, Quebec, Canada) and were grown and treated for 3 days with appropriate stimuli. Cells were rinsed twice with HBS, fixed with formaldehyde 3.7% (v/v) (in HBS), pH 7.4, for 15 min at room temperature, and permeabilized with 0.1% Triton X-100 (v/v) in HBS. Nonspecific binding was eliminated by washing with HBS/glycine (100 mM) solution for 1 h at 4°C followed by incubation with 5% milk solution. Cells were then incubated with the anti-tubulin antibody (1:50) for 1 h at room temperature. Coverslips were washed and further incubated for 1 h at room temperature with secondary fluorescein-conjugated anti-IgG antibody (Boehringer Mannheim). The coverslips were then mounted in Vectashield (Vector Laboratories, Burlingame, CA). We first examined the effects of Ang II on the morphological changes and cytoskeletal interactions that lead to the initial neurite outgrowth from neuronal cell bodies. Cells were plated at a density of 5 × 104 cells/dish. As shown by phase-contrast microscopy, after 3 days in culture, nontreated, nondifferentiated control cells were actively dividing and had round cell bodies, although some cells exhibited thin processes (Fig. 2A). After a 3-day treatment with Ang II (100 nM), most cells extended one or two neurite processes with a growth cone at their tip (Fig. 2B) along with a longer process, while the cell body retained a rounded appearance. Moreover, the number of cells was lower than in control, nontreated cells (from 4.3 ± 0.8 × 106 cells/control dishes to 3.2 ± 0.6 × 106 cells/Ang II-treated dishes, 25% decrease), indicating that the rate of cell division had decreased. Cell viability was not affected since they remained attached to the substratum, persistently excluded trypan blue, and the effect was reversible after removal of the hormone (data not shown). These morphological changes were due to AT2 receptor activation, since cells co-incubated with Ang II plus the AT2 receptor antagonist, PD 123319, kept their rounded appearance and formed aggregates (Fig. 2C), while co-incubation with Ang II plus DUP 753 did not alter the morphological appearance of Ang II-treated cells (data not shown). In addition, 3-day treatment with CGP 42112 also induced neurite extension and branching. These treated cells had a polygonal cell body, and the number of extensions and branchings appeared higher than in Ang II-treated cells (Fig. 2D). The morphological appearance of the cells after the 3-day treatment with Ang II was similar to those observed after a 3-day treatment with 1 mM dbcAMP, although in the latter, the cells had mainly one axon-like process and one or two dendrite-like processes (Fig. 3A). However, if Ang II was applied during the 3-day differentiating period, neurite retraction was observed, cells exhibit then a round up appearance but conserve one process (Fig. 3B). This morphological change was mediated by the AT1 receptor activation, since cells co-incubated with DUP 753 kept their differentiated morphology (Fig. 3C), as cells treated with CGP 42112 (Fig. 3D). Related changes in the distribution of microtubules were determined by immunofluorescence microscopy with an antibody to β-tubulin. In nondifferentiated cells, microtubules appear as long and thin filaments, loosely distributed throughout the cell (Fig. 4A). After a 3-day treatment with Ang II, most cells exhibited neurite processes with growth cones at their tips (Fig. 4B). When examined at higher magnification, intensification of microtubule labeling was noticed at the periphery of the cell body (Fig. 4C). Moreover, microtubules became organized in parallel bundles in the neurite and remained dispersed in the central part of the growth cone (Fig. 4D). In cells differentiated by a 3-day treatment with dbcAMP, microtubules were organized in longitudinal bundles localized within the cytoplasm of neurites and were abundant at the perinuclear region (Fig. 5A). When Ang II was added during the 3-day of differentiating period, cells became polygonal with microtubules retracted within the cell body (Fig. 5B). In order to quantify and to better characterize the pharmacology of the effects mediated by Ang II, the amount of polymerized tubulin was analyzed in detergent-extracted cytoskeletal fractions prepared from control, Ang II- or Ang II/analog-treated cells. Since neurite outgrowth requires large amounts of tubulin, its measurement represents an index of neurite extension. Cytoskeletal fractions from equivalent numbers of cells were analyzed in parallel. Thus, the cellular contents of each of the major proteins could be compared directly. As shown in Fig. 6A, in nondifferentiated cells treated for 3 days with Ang II, the level of tubulin had increased compared with nontreated control cells (lane 2 versus lane 1). The effect was specific to the AT2 receptor, since co-incubation with 10 µM PD 123319, a specific AT2 antagonist, reversed the effect of Ang II (lane 3 versus lane 1), whereas co-incubation with 10 µM DUP 753, which inhibits AT1 receptor, did not alter tubulin levels induced by Ang II (lane 4 versus lane 2). Moreover, 3-day treatment with 100 nM CGP 42112 (an AT2 receptor agonist) produced an effect similar to Ang II (lane 5 versus lane 2). Comparative densitometric analysis from five different experiments are illustrated in Fig. 7A and show that Ang II and CGP 42112, respectively, induced a 7- and 6- fold increase of tubulin levels compared with nontreated control cells and conclusively demonstrate that the effect is mediated through the AT2 receptor. Changes in microtubule levels may be due to an increase in total cell tubulin content or may reflect increased polymerization of the tubulin pool. In order to verify this point, total tubulin levels were measured in cellular extracts. As shown in Fig. 6B, Ang II did not induce significant changes in the level of total tubulin content.Fig. 7Quantitative densitometric analysis of the nondifferentiated (A, B) and differentiated NG108-15 cells (C, D) were treated for three days with Ang II or Ang II/analogs and harvested as described under “Materials and Methods.” Cytoskeletal fractions from equivalent numbers of cells were resolved in 4-15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. □, control; , 100 nM Ang II; 100 nM Ang II + 1 µM DUP 753; ▦, 100 nM Ang II + 10 µM PD 123319; □, 100 nM CGP 42112. Blots were probed with an anti-tubulin antibody (A, B) or MAP2 (C, D) and analyzed by densitometry. For MAP2, only the 85-kDa band has been scanned. Data are mean ± S.E. of five independent experiments. *, p < 0.02; **, p < 0.001, difference compared with the control value.View Large Image Figure ViewerDownload (PPT) Since MAPs affect microtubule assembly, stability, and cross-linking in developing brain (22Matus A. Annu. Rev. Neurosci. 1988; 11: 29-44Google Scholar), MAP2 and tau levels were characterized and compared in nondifferentiated control, Ang II- and Ang II/analog-treated cells. A monoclonal antibody recognizing a common epitope in both adult (MAP2a and -b) and juvenile (MAP2c) (27Beaman-Hall C.M. Vallano M.L. J. Neurobiol. 1993; 24: 1500-1516Google Scholar, 30Weisshaar B. Doll D. Matus A. Development (Camb.). 1992; 116: 1151-1161Google Scholar) forms was used in Western blots to assess protein levels of MAP2c in actively dividing and in differentiated cells. Fig. 6C shows that the antibody to MAP2 revealed two bands with similar intensities of around 85 and 76 kDa. Three-day treatment with Ang II induced a 5.3-fold increase in both MAP2c bands (Fig. 6C, lane 2 versus lane 1 and Fig. 7B). This effect was reproduced with CGP 42112 (4.7-fold increase) (Fig. 6C, lane 5 versus lane 1 and Fig. 7B) and reversed when Ang II was co-incubated with PD 123319 (lane 4 versus lane 2). However, addition of DUP 753, the AT1 receptor antagonist, also inhibited the Ang II effect (Fig. 6C, lane 3 versus lane 2 and Fig. 7B). Tau was detected with the monoclonal antibody 5E2. In nondifferentiated cells, tau appeared as a single isoform with a molecular mass of 58 kDa (Fig. 6D). The level of tau was not significantly affected by treatment with Ang II or Ang II/analogs. As equal amount of cells were analyzed by Western blotting, we could observe that the level of tubulin had increased by 5-fold in differentiated control cells compared with nondifferentiated control cells (compared lanes 1 in Fig. 6, A and E, and Fig. 7, A and C). The addition of Ang II during the 3 days of differentiating treatment with dbcAMP induced a small, but not significant, increase in the level of tubulin. However, when the AT1 receptor was inactivated by co-incubation with DUP 753, a net increase in the level of polymerized tubulin was observed (Fig. 6E, lane 3 versus lane 1) (2.5-fold increase over control, n = 5, Fig. 7C), which appeared to be mediated by the AT2 receptor. This observation was confirmed in cells treated with CGP 42112, which also induced an increase in tubulin content (Fig. 6E, lane 5 versus lane 1) (2.8-fold increase over control, n = 5, Fig. 7C). When PD 123319 was added together with Ang II, tubulin content was similar to that seen in control cells (Fig. 6E, lane 4 versus lane 1). As seen in nondifferentiated cells, total tubulin levels in control or Ang II- or Ang II/analog-treated cells did not change over the experimental period as compared with control (Fig. 6F). In differentiated control cells, the 76-kDa form of MAP2c was predominant (Fig. 6G, lane 1) and had increased compared with the nondifferentiated control cells (Fig. 6C, lane 1). Addition of Ang II for 3 days increased the level of the 85-kDa form without change in the 76-kDa form (Fig. 6G, lane 2 versus lane 1 and Fig. 7D). Here again, these effects were mediated by the AT2 receptor subtype, since co-incubation with PD 123319 inhibits the increase in the level of the 85-kDa form of MAP2 (Fig. 6G, lane 4 versus lane 2) and addition of CGP 42112 increased it (Fig. 6G, lane 5 versus lane 1 and Fig. 7D). However, the addition of DUP 753 did not modify significantly the level of MAP2 that was seen in cells under Ang II treatment (Fig. 6G, lane 3 versus lane 2 and Fig. 7D). Differentiation of NG108-15 cells by dbcAMP was accompanied by an increase in the level of the 58-kDa form of tau (Fig. 6, H versus D, lane 1). A minor band of 36 kDa was also present (Fig. 6H). Treatment with Ang II or Ang II/analogs did not significantly modify the basal level of tau. The most important observation of our study is that Ang II, via the AT2 receptor, acts on the microtubule-associated-protein, MAP2, initiating neurite outgrowth in nondifferentiated, rounded, and dividing NG108-15 cells. The second point is that opposite effects were observed in cells differentiated with dbcAMP, in which both AT1 and AT2 receptors are present. This illustrates a control of Ang II-induced differentiation by a negative feedback mechanism whereby the process is induced by AT2 receptor and inhibited by time-dependent expression of the AT1 receptor. When cultured in the presence of 10% FBS, NG108-15 cells contain only AT2 receptors. Although these results differ from those of Tallant et al. (31Tallant E.A. Diz D.I. Khosla M.C. Ferrario C.M. Hypertension. 1991; 17: 1135-1143Google Scholar), the groups of Speth et al. (32Speth R.C. Mei L. Yamamura H.I. Pept. Res. 1989; 2: 232-239Google Scholar) and Carrithers et al. (33Carrithers M.D. Masuda S. Koide K.A. Weyhenmeyer J.A. Neuroscience Lett. 1992; 135: 45-48Google Scholar) also found that, in nondifferentiated cells, Ang II receptor is not linked to G protein (33Carrithers M.D. Masuda S. Koide K.A. Weyhenmeyer J.A. Neuroscience Lett. 1992; 135: 45-48Google Scholar) and does not stimulate inositol phosphate production (32Speth R.C. Mei L. Yamamura H.I. Pept. Res. 1989; 2: 232-239Google Scholar), two properties associated with the AT1 receptor activation, thus supporting that in nondifferentiated cells, Ang II receptors are not of the AT1 type. These nondifferentiated cells treated for 3 days with Ang II develop neurites; this stimulation of neurite extension is accompanied by an increase in tubulin polymerization. Pharmacological studies demonstrate that these effects are attributable to the AT2 receptor, since application of CGP 42112 produces the same effects as Ang II, while co-incubation with DUP 753 does not alter the Ang II effect. Similar morphological changes were obtained in PC12 cells treated for 3 days with the well known differentiating factor, nerve growth factor (34Chiou J.-Y. Westhead E.W. J. Neurochem. 1992; 59: 1963-1966Google Scholar, 35Wu Y.Y. Bradshaw R.A. J. Cell Biol. 1993; 121: 409-422Google Scholar). We did not observe significant changes in total tubulin content after a 3-day treatment with Ang II and/or agonist or antagonists (Fig. 6B), which indicates that the nondifferentiated cells contain a pool of tubulin sufficient to support neurite outgrowth, as reported previously by Ferreira and Caceres (36Ferreira A. Caceres A. J. Neurosci. 1991; 11: 392" @default.
• W2046906382 created "2016-06-24" @default.
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• W2046906382 creator A5054524808 @default.
• W2046906382 creator A5055155293 @default.
• W2046906382 creator A5063729626 @default.
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• W2046906382 date "1996-09-01" @default.
• W2046906382 modified "2023-10-12" @default.
• W2046906382 title "Angiotensin II Induction of Neurite Outgrowth by AT2 Receptors in NG108-15 Cells" @default.
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