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• W2085673586 abstract "Microexplant cultures from three-day-old rats were used to investigate whether angiotensin II (Ang II), through its AT1 and AT2 receptors, could be involved in the morphological differentiation of cerebellar cells. Specific activation of the AT2 receptor during 4-day treatment induced two major morphological changes. The first was characterized by increased elongation of neurites. The second change was cell migration from the edge of the microexplant toward the periphery. Western blot analyses and indirect immunofluorescence studies revealed an increase in the expression of neuron-specific βIII-tubulin, as well as an increase in expression of the microtubule-associated proteins tau and MAP2. These effects were demonstrated by co-incubation of Ang II with 1 μm DUP 753 (AT1 receptor antagonist) or with 10 nm CGP 42112 (AT2 receptor agonist) but abolished when Ang II was co-incubated with 1 μm PD 123319 (AT2 receptor antagonist), indicating that differentiation occurs through AT2 receptor activation and that the AT1 receptor inhibits the AT2 effect. Taken together, these results demonstrate that Ang II is involved in cerebellum development for both neurite outgrowth and cell migration, two important processes in the organization of the various layers of the cerebellum. Microexplant cultures from three-day-old rats were used to investigate whether angiotensin II (Ang II), through its AT1 and AT2 receptors, could be involved in the morphological differentiation of cerebellar cells. Specific activation of the AT2 receptor during 4-day treatment induced two major morphological changes. The first was characterized by increased elongation of neurites. The second change was cell migration from the edge of the microexplant toward the periphery. Western blot analyses and indirect immunofluorescence studies revealed an increase in the expression of neuron-specific βIII-tubulin, as well as an increase in expression of the microtubule-associated proteins tau and MAP2. These effects were demonstrated by co-incubation of Ang II with 1 μm DUP 753 (AT1 receptor antagonist) or with 10 nm CGP 42112 (AT2 receptor agonist) but abolished when Ang II was co-incubated with 1 μm PD 123319 (AT2 receptor antagonist), indicating that differentiation occurs through AT2 receptor activation and that the AT1 receptor inhibits the AT2 effect. Taken together, these results demonstrate that Ang II is involved in cerebellum development for both neurite outgrowth and cell migration, two important processes in the organization of the various layers of the cerebellum. angiotensin II 1,4-piperazinediethanesulfonic acid glial fibrillary acidic protein microtubule-associated protein A large number of studies indicate that the hormone angiotensin II (Ang II)1 and its receptors are present in the brain (1Lenkei Z. Palkovits M. Corvol P. Llorens-Cortes C. Front. Neuroendocrinol. 1997; 18: 383-439Crossref PubMed Scopus (355) Google Scholar, 2Ferguson A. Washburn D. Prog. Neurobiol. 1997; 54: 169-192Crossref Scopus (129) Google Scholar). As in the periphery, the AT1 receptor exhibits a high affinity for the nonpeptidic antagonist DUP 753 (Losartan), whereas the AT2 receptor has a high affinity for the antagonist PD 123319 and the agonist CGP 42112 (1Lenkei Z. Palkovits M. Corvol P. Llorens-Cortes C. Front. Neuroendocrinol. 1997; 18: 383-439Crossref PubMed Scopus (355) Google Scholar, 2Ferguson A. Washburn D. Prog. Neurobiol. 1997; 54: 169-192Crossref Scopus (129) Google Scholar). Although the AT1 receptors are detected in areas involved in the regulation of blood pressure, hydromineral balance, and thirst, no central function has yet been attributed to the AT2 receptor. This receptor is highly expressed in the inferior olive, locus coeruleus, thalamic nuclei, medial geniculate nuclei, and the molecular layer of the cerebellum (3Tsutsumi K. Saavedra J.M. Am. J. Physiol. 1991; 261: R209-R216PubMed Google Scholar, 4Jöhren O. Inagami T. Saavedra J. Neuroreport. 1995; 6: 2549-2552Crossref PubMed Scopus (102) Google Scholar, 5Lenkei Z. Palkovits M. Corvol P. Llorens-Cortes C. J. Comp. Neurol. 1996; 373: 322-339Crossref PubMed Scopus (76) Google Scholar). Although several studies have been conducted on the short term effect of AT2 receptor activation on intracellular events, a few studies focused on the physiological function of the AT2receptor. One well described function is its antagonistic action on cellular growth induced by neurotrophic factors (nerve growth factor) (6Tsuzuki S. Eguchi S. Inagami T. Biochem. Biophys. Res. Commun. 1996; 228: 825-830Crossref PubMed Scopus (66) Google Scholar, 7Meffert S. Stoll M. Steckelings U.M. Bottari S.P. Unger T. Mol. Cell. Endocrinol. 1996; 122: 59-67Crossref PubMed Scopus (208) Google Scholar) or by the AT1 receptor of Ang II (8Stoll M. Steckelings M. Paul M. Bottari S. Metzger R. Unger T. J. Clin. Invest. 1995; 95: 651-657Crossref PubMed Google Scholar, 9Nakajima M. Hutchinson H. Fujinaga M. Hayashida W. Morishita R. Zhang L. Horiuchi M. Pratt R. Dzau V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10663-11667Crossref PubMed Scopus (649) Google Scholar, 10Munzenmaier D. Greene A. Hypertension. 1996; 27: 760-765Crossref PubMed Google Scholar). Another function recently described for the AT2 receptor is a role in programmed cell death (11Tanaka M. Ohnishi J. Ozawa Y. Sugimoto M. Usuki S. Naruse M. Murakami K. Miyazaki H. Biochem. Biophys. Res. Commun. 1995; 207: 593-598Crossref PubMed Scopus (124) Google Scholar, 12Yamada T. Horiuchi M. Pratt R. Dzau V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 156-160Crossref PubMed Scopus (665) Google Scholar). Interestingly, although the expression of the AT1 receptor either remains stable or increases with development in rats, the expression and density of the AT2 receptor decrease dramatically with maturation from fetal to neonatal to adult, both at the periphery and in several brain nuclei (13Millan M.A. Jacobowitz D.M. Aguilera G. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11440-11444Crossref PubMed Scopus (182) Google Scholar, 14Tsutsumi K. Strömberg C. Viswanathan M. Saavedra J.M. Endocrinology. 1991; 129: 1075-1082Crossref PubMed Scopus (135) Google Scholar, 15Schütz S. Le Moullec J.-M Corvol P. Gasc J.-M. Am. J. Pathol. 1996; 149: 2067-2079PubMed Google Scholar, 16Breault L. Lehoux J.-G. Gallo-Payet N. J. Clin. Endocrinol. Metab. 1996; 81: 3914-3922PubMed Google Scholar). This high and transient expression of the AT2 receptor in fetal tissues suggests that it may play a specific role during development and cellular differentiation (7Meffert S. Stoll M. Steckelings U.M. Bottari S.P. Unger T. Mol. Cell. Endocrinol. 1996; 122: 59-67Crossref PubMed Scopus (208) Google Scholar, 11Tanaka M. Ohnishi J. Ozawa Y. Sugimoto M. Usuki S. Naruse M. Murakami K. Miyazaki H. Biochem. Biophys. Res. Commun. 1995; 207: 593-598Crossref PubMed Scopus (124) Google Scholar,17Yoshimura Y. Karube M. Aoki H. Oda T. Koyama N. Nagai A. Akimoto Y. Hirano H. Nakamura Y. Endocrinology. 1996; 137: 1204-1211Crossref PubMed Scopus (81) Google Scholar, 18Laflamme L. De Gasparo M. Gallo J.-M. Payet M.D. Gallo-Payet N. J. Biol. Chem. 1996; 271: 22729-22735Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Indeed, in a previous study, using neuroblastoma × glioma hybrid NG108–15 cells, we have shown that a 3-day treatment with Ang II or CGP 42112 induced neurite outgrowth characterized by an increase in the level of polymerized tubulin and in the association of the microtubule-associated protein MAP2c with microtubules (18Laflamme L. De Gasparo M. Gallo J.-M. Payet M.D. Gallo-Payet N. J. Biol. Chem. 1996; 271: 22729-22735Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). A similar effect was observed in PC12W cells, with a decrease in another microtubule-associated protein, MAP1B (7Meffert S. Stoll M. Steckelings U.M. Bottari S.P. Unger T. Mol. Cell. Endocrinol. 1996; 122: 59-67Crossref PubMed Scopus (208) Google Scholar), and an increase in the association of MAP2 with microtubules (19Stroth U. Meffert S. Gallinat S. Unger T. Mol. Brain. Res. 1998; 53: 187-195Crossref PubMed Scopus (70) Google Scholar) as well as with an increase in the neurofilament middle subunit NF-M (20Gallinat S. Csikos T. Meffert S. Herdegen T. Stoll M. Unger T. Neurosci. Lett. 1997; 9: 29-32Crossref Scopus (47) Google Scholar). Neuronal development and differentiation of the cerebellum involve several steps including proliferation (in the ventricular zone), migration (through the ventricular zone to the cortical zone), and finally either neurite extension or apoptosis, once the cells have reached their specific location (21Cambray-Deakin M. Morgan A. Burgoyne R. Dev. Brain Res. 1987; 37: 197-207Crossref Scopus (28) Google Scholar, 22Komuro H. Rakic P. J. Neurosci. 1998; 15: 1478-1490Crossref Google Scholar). Each of these steps is controlled by several local environmental cues, such as components of the extracellular matrix and cell adhesion molecules (23Rocamora N. Garcia-Ladona F. Palacios J. Mengod G. Mol. Brain Res. 1993; 17: 1-8Crossref PubMed Scopus (156) Google Scholar, 24Goldowitz D. Hamre K. Trends Neurosci. 1998; 21: 375-382Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). However, it is clear that the molecular identity of all of the regulators is yet to be determined. In particular, despite the studies conducted on neuronal cell lines (see above), so far there have been no studies on the long term effect of Ang II on either neuronal differentiation or neuronal development of specific brain areas. Therefore, the aim of the present study was to investigate the role of the AT1 and AT2 receptors on neurite outgrowth and on the pattern of expression of tubulin as well as of tau and MAP2, two important microtubule-associated proteins, in primary cultures of neonatal rat cerebellar neurons. The cerebellum was chosen for several reasons: 1) Ang II as well as both AT1 and AT2 receptors are present throughout cerebellar development (3Tsutsumi K. Saavedra J.M. Am. J. Physiol. 1991; 261: R209-R216PubMed Google Scholar, 14Tsutsumi K. Strömberg C. Viswanathan M. Saavedra J.M. Endocrinology. 1991; 129: 1075-1082Crossref PubMed Scopus (135) Google Scholar, 25Jöhren O. Saavedra J. Neuroreport. 1996; 7: 1349-1352Crossref PubMed Scopus (15) Google Scholar, 26Jöhren O. Häuser W. Saavedra J. Brain Res. 1998; 793: 176-186Crossref PubMed Scopus (14) Google Scholar), suggesting a role for Ang II in this brain structure (26Jöhren O. Häuser W. Saavedra J. Brain Res. 1998; 793: 176-186Crossref PubMed Scopus (14) Google Scholar, 27Erdmann B. Fuxe K. Ganten D. Hypertension. 1996; 28: 818-824Crossref PubMed Scopus (68) Google Scholar). 2) Differentiation of cerebellar granule cells occurs mostly during the postnatal period (21Cambray-Deakin M. Morgan A. Burgoyne R. Dev. Brain Res. 1987; 37: 197-207Crossref Scopus (28) Google Scholar, 28Burgoyne R. Cambray-Deakin M. EMBO J. 1988; 7: 2311-2319Crossref PubMed Scopus (140) Google Scholar), implicating both cell migration from the molecular layer to the Purkinje cell layer and neurite elongation (22Komuro H. Rakic P. J. Neurosci. 1998; 15: 1478-1490Crossref Google Scholar). 3) The juvenile and adult forms of tau and MAP2 are differentially expressed during maturation of this brain area and may serve as markers of the state of neuronal maturation (29Binder L.I. Kim H. Caceres A. Payne M.R. Rebhun L.I. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5613-5617Crossref PubMed Scopus (197) Google Scholar, 30Burgoyne R. Cumming R. Neuroscience. 1984; 11: 157-167Crossref Scopus (97) Google Scholar). The chemicals used in the present study were obtained from the following sources. Angiotensin II was from Bachem (Marina Delphen, CA); glutamine, neurobasal medium, and B27 supplement were from Life Technologies, Inc. CGP 42112, DUP 753, and PD 123319 were synthesized at Ciba-Geigy, Basel, Switzerland), and anti-mouse IgG-fluorescein was from Amersham Pharmacia Biotech. Monoclonal anti-β-tubulin and enhanced chemiluminescence detection system were from Roche Molecular Biochemicals; anti-GFAP, anti-neurofilament NE-14, anti-βIII-tubulin, and the monoclonal antibody HM-2, which recognizes all MAP2 isoforms, were purchased from Sigma. Monoclonal tau antibody 5E2 was kindly provided by Dr. Kenneth Kosik (Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA), and tau 1 antibody was kindly provided by Dr. Lester Binder (Department of Cell Biology, University of Alabama at Birmingham, AL). Ang II was iodinated in the laboratory of Dr. Gaetan Guillemette (Department of Pharmacology, University of Sherbrooke, PQ, Canada). Vectashield mounting medium was from Vector Laboratories (Berlingame, CA). All other chemicals were of A grade purity. Primary cultures of mixed cerebellar cells were prepared from the methodology described by Moonenet al. (31Moonen G. Neale E. Macdonald R. Gibbs W. Nelson P. Brain Res. 1982; 281: 59-73Crossref PubMed Scopus (34) Google Scholar), with the following modifications. Cerebelli (10–12/culture) from Long Evans rats at postnatal day 3 were isolated and mechanically dissociated in neurobasal medium supplemented with B27 and 0.5 mm glutamine. The suspension was centrifuged at 100 × g for 10 min at room temperature. The cell pellet was suspended in the same medium and plated at a density of 1.5 × 106 cells/35-mm Petri dish, precoated with poly-l-lysine. Cells were grown in a humidified atmosphere of 95% air, 5% CO2, at 37 °C. 24 h after plating, cells were treated for 4 consecutive days without (control cells) or with CGP 42112, the AT2 receptor agonist (10 nm), or with Ang II (100 nm) alone or in the presence of DUP 753, an AT1 receptor antagonist (1 μm), or PD 123319, an AT2 receptor antagonist (1 μm) and were used on the 6th day. Ang II binding studies were conducted on cells cultured for 6 days according to the methodology previously described (18Laflamme L. De Gasparo M. Gallo J.-M. Payet M.D. Gallo-Payet N. J. Biol. Chem. 1996; 271: 22729-22735Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Density of Ang II receptor subtypes were identified as total binding sensitivity toward the AT1 and AT2 receptor analogs. Preparations enriched in microtubules were obtained from cells cultured for 6 days in 35-mm Petri dishes as described by Solomon (32Solomon F. Methods Enzymol. 1986; 134: 139-147Crossref PubMed Scopus (33) Google Scholar) with slight modifications described previously (18Laflamme L. De Gasparo M. Gallo J.-M. Payet M.D. Gallo-Payet N. J. Biol. Chem. 1996; 271: 22729-22735Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). 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 (0.1 m PIPES, 2 mglycerol, 5 mm MgCl2, 2 mm EGTA, 0.04 trypsin inhibitor unit/ml aprotinin, 2 mmphenylmethylsulfonyl fluoride, 1 mm benzamidine, pH 6.9) containing Taxol (1 μm). After collection and centrifugation (1,000 × g for 5 min at 37 °C), the cell pellet was extracted with PM2G buffer containing 1% Nonidet P-40 and 1 μm Taxol (15 min incubation at 37 °C). After centrifugation, the resulting pellet (containing microtubules) was solubilized in electrophoresis sample buffer (Tris buffer 62.5 mm, pH 6.8 containing 2% SDS (w/v), 10% glycerol (v/v), 5% β-mercaptoethanol) and heated to 100 °C for 5 min. After centrifugation (10,000 × g for 5 min), the supernatant was stored at −20 °C. For total cell extracts, cells grown in 35-mm Petri dishes were washed twice with HBS buffer 13. mm NaCl, 3.5 mm KCl, 1.8 mm CaCl2, 0.5 mm MgCl2, 2.5 mmNaHCo3, 5 mm HEPES, scraped, and solubilized as described above. Heat-stable cytoplasmic extracts were prepared as described previously (33Smith C. Anderton B. Davis D. Gallo J. FEBS Lett. 1995; 375: 243-248Crossref PubMed Scopus (72) Google Scholar). Briefly, cells were scraped in HBS, immediately boiled for 5 min, and centrifugated (20,000 × g for 20 min). Supernatants containing MAPs were diluted 1:1 in 2× sample buffer and heated to 100 °C for 5 min. Brain preparations enriched in MAPs were also obtained from postnatal day 13 and adult rats. Samples from equivalent number of cells were compared in each experiment. Samples were separated on 8% SDS-polyacrylamide gels. Proteins were transferred electrophoretically to polyvinylidene difluoride membranes (Roche Molecular Biochemicals). Membranes were blocked with 1% gelatin, 0.05% Tween 20 in TBS buffer (pH 7.5). After washing with TBS-Tween 20 (0.05%), membranes were incubated overnight at 4 °C with anti-β-tubulin (1:500), anti-βIII-tubulin (1:400), tau 1 antibody (1:1000), 5E2 antibody (1:1000), or HM-2 antibody (1:500), diluted in TBS-Tween 20 (0.05%) plus bovine serum albumin (0.1%). After washing with TBS-Tween 20, detection was accomplished using horseradish peroxidase-conjugated anti-mouse IgG (1:2000) and an enhanced chemiluminescence detection system. Immunoreactivity was quantified by densitometry with ImageQuant software and expressed as arbitrary units. The anti-GFAP antibody was used as glial marker, whereas NE-14 and anti-βIII-tubulin antibodies were used as neuronal markers. Cells were washed twice with HBS, then fixed with methanol for 10 min at −20 °C. Cells were then rehydrated and incubated with anti-βIII-tubulin (1:40), anti-GFAP (1:20), and NE-14 (1:20) antibodies for 1 h at room temperature. After washing, cells were incubated (1 h at room temperature) with an anti-mouse IgG coupled to fluorescein isothiocyanate (1:30). After washing, slides were mounted in Vectashield and examined on a Nikon DM 400 microscope equipped for epifluorescence using a B-1E fluorescein isothiocyanate filter set (Nikon, Melville, NY). To determine cell migration, nuclear DNA staining was performed using propidium iodine as described previously (34Johnson J. Methods Cell Biol. 1995; 46: 243-276Crossref PubMed Scopus (28) Google Scholar) with slight modifications. Cells were washed twice with HBS buffer, fixed with methanol (10 min at −20 °C), rehydrated (for 10 min), and incubated with propidium iodine (1 μg/ml) for 20 min at room temperature. After washing, slides were examined using a G-2A orange range filter set. To quantify cell migration, 12 consecutive rings (multiple of approximately 50 μm), beginning at the edge of the microexplant, were drawn. The distances traveled by the cells were defined as the percentage of cells located in a precise ring over the total number of cells from the first to the last ring. The percentage of cells that had exhibited migration was calculated as the total number of cells located from the second (first stage of migration) to the last ring, over the total number of cells. The data are presented as the means ± S.E. Statistical analyses of the data were performed using Studentt test, and p values were obtained from Dunnett's tables. n indicates the number of experiments, each performed in triplicate. As reported previously, cerebellar cells survive and differentiate when cultured as microexplants in conditioned serum-free medium. 24 h after seeding, small clusters of cells exhibited short processes, which increased in number and length as the culture period progressed. After 6 days, cells exhibit several long radially oriented neurites extended from the microexplant. Indirect immunofluorescence studies, using neuronal and glial markers indicated that the cell cultures were composed of approximately 50% neurons and 50% glial cells (Fig. 1). Co-incubation of Ang II with AT1 or AT2 receptor analogs indicated that the cell cultures contained both AT1 and AT2receptor subtypes (Fig. 2).Figure 2Angiotensin II binding in cerebellar microexplant cultures. After 6 days in culture, [125I]Sar1-Ile8-Ang II bindingTot was determined as described (18Laflamme L. De Gasparo M. Gallo J.-M. Payet M.D. Gallo-Payet N. J. Biol. Chem. 1996; 271: 22729-22735Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Nonspecific binding (NS) was determined after incubation in the presence of 1 μm unlabeled Ang II. Binding in the presence of 1 μm DUP 753 (DUP) or CGP 42112 (CGP) indicates proportion of AT1 and AT2 receptors. Results are expressed as fmol/mg membrane proteins. Results are the means ± S.E. of three experiments, each in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Four-day treatment of cerebellar microexplant cultures with 100 nm Ang II induced important morphological changes. Two major modifications were observed. The first involved an increase in length of processes that exhibited several arborizations, and second, a marked cell migration was observed from the edge of the microexplant toward the periphery (Fig. 3,B compared with A). These effects were more pronounced in cells treated with Ang II and DUP 753 (1 μm), a specific AT1 receptor antagonist (Fig.3 C) or in cells treated with 10 nm of CGP 42112, the AT2 receptor agonist (Fig. 3 E). Moreover, incubation with Ang II and PD 123319 (1 μm), a specific antagonist of the AT2 receptor, blocked the AT2receptor-mediated effect (Fig. 3 D). Alone, DUP 753 and PD 123319 did not affect cell morphology compared with control cells, whereas PD 123319 abolished the effect of CGP 42112 (data not shown). Two protocols were used to quantify the morphological changes induced by Ang II treatment. Because process elongation involves an increase in the polymerization of tubulin, we measured the level of tubulin incorporated into cytoskeletal fractions from control, Ang II-treated, and Ang II/analog-treated cells. As shown in Fig.4, Ang II alone did not significantly modify the level of polymerized tubulin. However, specific stimulation of the AT2 receptor through either incubation of CGP 42112 or co-incubation with Ang II and DUP 753 increased the level of polymerized tubulin by 1.67 ± 0.15- and 1.40 ± 0.18-fold, respectively (n = 3). On the other hand, stimulation of the AT1 receptor by co-incubating Ang II with PD 123319 abolished the AT2 receptor-mediating effect (Fig.4 A). However, when incubated alone, DUP 753 or PD 123319 did not modify the basal level of polymerized tubulin, whereas PD 123319 abolished the effect of CGP 42112 (Fig. 4 B). By comparison, Ang II and/or analogs did not change the total level of tubulin content (Fig. 4 C). To evaluate whether process elongation affected neurons or glial cells, the effect of Ang II and analogs was verified on immunofluorescence of βIII-tubulin, an isoform specifically localized in neurons (35Ferreira A. Caceres A. J. Neurosci. Res. 1992; 32: 516-529Crossref PubMed Scopus (93) Google Scholar). As shown in Fig.5, AT2 receptor activation clearly increased the level of βIII-tubulin labeling associated with neurons. After 4 days of treatment with CGP 42112 (Fig. 5 E) or Ang II plus DUP 753 (Fig. 5 C), the cells had a well developed network of neurites with several varicosities. In these conditions, processes appeared thicker, suggesting that AT2receptor activation increased fasciculation. Again, morphological observations were correlated with Western blot analyses (Fig.6) and confirmed that AT2receptor activation increased the level of βIII-tubulin incorporated into microtubules (Fig. 6, A and B), an effect abolished by the addition of PD 123319 but without any effect on the total level of βIII-tubulin in cells (Fig. 6 C).Figure 5Immunofluorescence microscopy analysis of the effect of Ang II on βIII-tubulin in cerebellar microexplant cultures. Microexplants were cultured and stimulated for 4 days alone (A) or with Ang II (B), Ang II + DUP 753 (C), Ang II + PD 123319 (D), or CGP 42112 (E) as explained in the legend to Fig. 3. After methanol fixation, cells were processed for immunofluorescence labeling using an anti-βIII-tubulin (1:40 dilution) and anti-mouse IgG-fluorescein isothiocyanate as described under “Experimental Procedures.” Thebars represent 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Western blot analysis of the effect of angiotensin II on the level of polymerized βIII-tubulin in cerebellar microexplant cultures. Microexplants were cultured and stimulated for 4 days as a control (bar C) or with Ang II, Ang II + DUP 753 (+DUP), Ang II + PD 123319 (+PD), or CGP 42112 (CGP) as explained in the legend to Fig. 3. A, quantitative densitometric analysis of the effect of Ang II on the level of polymerized βIII-tubulin. Data are the means ± S.E. of three independent experiments. B, representative Western immunoblotting of polymerized tubulin performed on microtubule-enriched preparations. C, representative Western immunoblotting of polymerized βIII-tubulin performed on whole cell extracts (total βIII-tubulin level). **, p < 0.001, difference compared with control value. Numbers on the leftindicate the molecular masses (kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Synthesis of MAPs represents critical events during elaboration of neurites. Several studies indicate that this synthesis follows a time course that is correlated with axonal and dentritic growth (36Matus A. Annu. Rev. Neurosci. 1988; 11: 29-44Crossref PubMed Scopus (513) Google Scholar). We therefore studied whether Ang II could affect the level of expression of tau and MAP2, two MAPs specifically expressed in axons and dendrites, respectively (36Matus A. Annu. Rev. Neurosci. 1988; 11: 29-44Crossref PubMed Scopus (513) Google Scholar, 37Avila J. Dominguez J. Diaz-Nido J. Int. J. Dev. Biol. 1994; 38: 13-25PubMed Google Scholar). Tau 1 antibody identified a group of several tau isoforms. A representative Western blot in Fig.7 A illustrates that AT2 receptor activation by Ang II, after inhibition of the AT1 receptor by DUP 753 or after CGP 42112 treatment, strongly increased the level of the tau protein. In control cells, a single band of 50 kDa was observed, whereas in Ang II plus DUP 753- or in CGP 42112-treated cells, isoforms of higher molecular weight began to appear. The far right lane in Fig. 7 A shows the control pattern of tau expression in brain extracts from postnatal day 13 and from adult rats. As expected, a single isoform was detected in the young rat, whereas several bands were revealed in the adult (36Matus A. Annu. Rev. Neurosci. 1988; 11: 29-44Crossref PubMed Scopus (513) Google Scholar, 37Avila J. Dominguez J. Diaz-Nido J. Int. J. Dev. Biol. 1994; 38: 13-25PubMed Google Scholar, 38Brion J. Smith C. Couck A. Gallo J. Anderton B. J. Neurochem. 1993; 61: 2071-2080Crossref PubMed Scopus (170) Google Scholar, 39Przyborski S. Cambray-Deakin M. Dev. Brain Res. 1995; 89: 187-201Crossref PubMed Scopus (41) Google Scholar, 40Przyborski S. Cambray-Deakin M. Dev. Brain Res. 1995; 87: 29-45Crossref PubMed Scopus (10) Google Scholar). Parallel Western blots with the 5E2 antibody, which recognizes unphosphorylated and phosphorylated forms of tau, revealed a stronger effect of Ang II, via the AT2 receptor, suggesting that the difference with the tau 1 immunoblot (Fig.7 A) is due to a preferential increase in tau phosphorylation. Fig. 7 (C and D) illustrates the effect of Ang II and analogs on the level of HMW-MAP2 and on MAP2c. Adult HMW-MAP2 was resolved in two isoforms, termed MAP2a and MAP2b of 280 kDa; MAP2a is present early in development, whereas the expression of MAP2c disappears in the adult. Again, AT2 receptor stimulation increased the levels of both proteins. As for tubulin measurements, incubation with DUP 753 or PD 123319 alone exhibited the same immunoreactivity against tau and MAP 2 than control cells (data not shown). Quantification of cell migration was performed as described under “Experimental Procedures” following cell nuclei labeling with propidium iodine as shown in Fig. 8. Results from Fig. 8 A indicate that under CGP 42112 treatment, cells exhibited the highest degree of cell migration. Measurement of the number of migrating cells from the edge of the microexplant to the outermost peripheral ring indicates that Ang II induced a 1.9 ± 0.1-fold increase (n = 3) in the number of migrating cells, compared with control cultures. In corroboration with microscopic examination, these effects were due to AT2 receptor activation, because CGP 421122 or Ang II plus DUP 753 induced a stronger effect (3.3 ± 0.21- and 3.1 ± 0.25-fold, respectively, n = 3), whereas co-incubation with PD 123319 blocked these effects (1.2 ± 0.21-fold difference compared with control, n = 3) (Fig.9).Figure 9Quantification of the effect of Ang II on cell migration in cerebellar microexplant cultures. A, cell migration was quantified as defined under “Experimental Procedures” by counting the number of cells in a particular ring over the total number of cells from the first to the last ring of migration. Representative analysis for one microexplant is shown. B, quantitative analysis of the percentage of cells that had exhibited migration, calculated as the total number of cells located from the second ring (first stage of migration) to the last ring, over the total number of cells. Results represent the means ± S.E. of three experiments, with a minimum of four microexplants analyzed for each experiment. **, p < 0.001, difference compared with control value.View Large" @default.
• W2085673586 created "2016-06-24" @default.
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• W2085673586 creator A5054524808 @default.
• W2085673586 creator A5063729626 @default.
• W2085673586 creator A5080932342 @default.
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• W2085673586 date "1999-10-01" @default.
• W2085673586 modified "2023-10-17" @default.
• W2085673586 title "Activation of the AT2 Receptor of Angiotensin II Induces Neurite Outgrowth and Cell Migration in Microexplant Cultures of the Cerebellum" @default.
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