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• W1992807239 abstract "Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40–50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca2+, a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca2+]i transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo [Ca2+]i increase upon specific stimuli is present in cultured hippocampal astrocytes. Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40–50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca2+, a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca2+]i transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo [Ca2+]i increase upon specific stimuli is present in cultured hippocampal astrocytes. Astrocytes make up a large percentage of the cell composition of the central nervous system (CNS) 1The abbreviations used are: CNS, central nervous system; SgII, secretogranin II; CgA and B, chromogranin A and B; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; GFAP, glial fibrillary acidic protein; PNS, post-nuclear supernatants; DMEM, Dulbecco's modified Eagle's medium 1The abbreviations used are: CNS, central nervous system; SgII, secretogranin II; CgA and B, chromogranin A and B; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; GFAP, glial fibrillary acidic protein; PNS, post-nuclear supernatants; DMEM, Dulbecco's modified Eagle's medium and are thought to be involved in many important brain functions. Increasing evidence indicates that these cells can interact with the surrounding neurons and exhibit the equipment to receive, integrate, and transmit signals. It has been reported that astrocytes express a number of membrane ionic channels, transporters, and receptors linked to the most important signal transduction pathways and are capable of intracellular propagation of slow calcium waves (1Cornell-Bell A.H. Finkbeiner S.M. Cooper M.S. Smith S.J. Science. 1990; 247: 470-473Crossref PubMed Scopus (1404) Google Scholar, 2Charles A.C. Merrill J.E. Dirksen E.R. Sanderson M.J. Neuron. 1991; 6: 983-992Abstract Full Text PDF PubMed Scopus (542) Google Scholar, 3Sontheimer H. Black J.A. Waxman S.G. Trends Neurosci. 1996; 19: 325-331Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 4Steinhauser C. Gallo V. Trends Neurosci. 1996; 19: 339-345Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 5Verkhratsky A. Kettenmann H. Trends Neurosci. 1996; 19: 346-352Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 6Bacci A. Verderio C. Pravettoni E. Matteoli M. Philos. Trans. R. Soc. Lond-Biol. Sci. 1999; 354: 403-409Crossref PubMed Scopus (80) Google Scholar). Furthermore, astrocytes may secrete different regulatory molecules, neurotrophic factors, and neuropeptides. In situ hybridization and immunohistochemical studies have revealed the presence of proenkephalin in cultured as well as in rat brain astrocytes (7Shinoda H. Marini A.M. Cosi C. Schwartz J.P. Science. 1989; 245: 415-417Crossref PubMed Scopus (188) Google Scholar, 8Hauser K.F. Osborn J.G. Stiene-Martin A. Melner M.H. Brain Res. 1990; 522: 347-353Crossref PubMed Scopus (73) Google Scholar, 9Spruce B.A. Curtis R. Wilkin G.P. Glover D.M. EMBO J. 1990; 9: 1787-1795Crossref PubMed Scopus (94) Google Scholar, 10Klein R.S. Fricker L.D. Brain Res. 1992; 569: 300-310Crossref PubMed Scopus (37) Google Scholar). Other regulatory (poly)peptides (somatostatin, cholecystokinin) and neuropeptide processing enzymes (carboxypeptidase E and peptidylglycine-α-amidating mono-oxygenase), which are known to be co-stored in secretory granules (11.Mains, R. E., Cullen, E. I., May, V., and Eipper, B. A.Ann. N. Y. Acad. Sci. 493, 278–291.Google Scholar, 12Halban P.A. Irminger J.C. Biochem. J. 1994; 299: 1-18Crossref PubMed Scopus (278) Google Scholar), have been detected in astrocytes (10Klein R.S. Fricker L.D. Brain Res. 1992; 569: 300-310Crossref PubMed Scopus (37) Google Scholar, 13Vilijn M.H. Das B. Kessler J.A. Fricker L.D. J. Neurochem. 1989; 53: 1487-1493Crossref PubMed Scopus (39) Google Scholar, 14Klein R.S. Fricker L.D. Brain Res. 1992; 596: 202-208Crossref PubMed Scopus (14) Google Scholar). In addition, it has been shown that cultured astrocytes and Bergmann glial cells express secretogranin II (SgII) and chromogranin A (CgA), respectively (15Ficher-Colbrie R. Kirchmair R. Schobert A. Olenik C. Meyer D.K. Winkler H. J. Neurochem. 1993; 60: 2312-2314Crossref PubMed Scopus (26) Google Scholar,16McAuliffe W.G. Hess A. GLIA. 1990; 3: 13-16Crossref PubMed Scopus (11) Google Scholar), two well characterized members of the granin family (for reviews, see Refs. 17Huttner W.B. Gerdes H.H. Rosa P. Trends Biochem. Sci. 1991; 16: 27-30Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 18Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (609) Google Scholar, 19Rosa P. Gerdes H.H. J. Endocrinol. Invest. 1994; 17: 207-225Crossref PubMed Scopus (105) Google Scholar). Besides the discovery of their presence, little information is available, however, on the subcellular localization and the role of SgII and CgA in glial cells. Like the well established neuropeptides and hormones, granins are known to be stored in the dense-core secretory granules of many neuroendocrine cells (18Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (609) Google Scholar, 19Rosa P. Gerdes H.H. J. Endocrinol. Invest. 1994; 17: 207-225Crossref PubMed Scopus (105) Google Scholar). However, the distribution of the individual members of the granin family is more widespread than that known for any other neuropeptide or polypeptide hormone. Furthermore, the granins were detected in dense-core vesicles of endocrinologically silent neuroendocrine tumors such as the nonfunctioning pituitary adenomas (20Rosa P. Bassetti M. Weiss U. Huttner W.B. J. Histochem. Cytochem. 1992; 40: 523-533Crossref PubMed Scopus (31) Google Scholar), thus suggesting that they may be sufficient for the formation of the dense-matrix of secretory granules. Given their widespread distribution, the granins have been used as the most useful markers to investigate the presence of dense-core granules (also referred to as large dense-core vesicles) in neurons of different areas of the mammalian CNS (18Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (609) Google Scholar, 19Rosa P. Gerdes H.H. J. Endocrinol. Invest. 1994; 17: 207-225Crossref PubMed Scopus (105) Google Scholar, 21Scamell J.G. Trends Endocrinol. Metab. 1993; 4: 14-18Abstract Full Text PDF PubMed Scopus (18) Google Scholar). Consistent with their subcellular localization, the granins are widely investigated in vivo and in vitro with the aim of studying the mechanisms of secretory granule biogenesis (22Rosa P. Weiss U. Pepperkok R. Ansorge W. Niehrs C. Stelzer E.H. Huttner W.B. J. Cell Biol. 1989; 109: 17-34Crossref PubMed Scopus (82) Google Scholar, 23Tooze S.A. Huttner W.B. Cell. 1990; 60: 837-847Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 24Chanat E. Huttner W.B. J. Cell Biol. 1991; 115: 1505-1519Crossref PubMed Scopus (383) Google Scholar). This process can be divided into two major steps: (i) the selective sorting of regulated secretory proteins from other proteins destined for different pathways and (ii) the budding from the trans-Golgi network of the dense-core vesicles (12Halban P.A. Irminger J.C. Biochem. J. 1994; 299: 1-18Crossref PubMed Scopus (278) Google Scholar, 25Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (737) Google Scholar, 26Bauerfeind R. Huttner W.B. Curr. Opin. Cell Biol. 1993; 5: 628-635Crossref PubMed Scopus (133) Google Scholar). These vesicles are then stored intracellularly until a specific signal evokes their exocytosis. The regulated release of neurotransmitters and regulatory polypeptides from neuronal and neuroendocrine cells has been shown to depend on molecular interactions between integral membrane proteins of the neurosecretory vesicles (v-SNARE) and the plasma membrane (t-SNARE) (for reviews see Refs. 27Scheller R.H. Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 28Goda Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 769-772Crossref PubMed Scopus (57) Google Scholar, 29Hanson P.I. Heuser J.E. Jahn R. Curr. Opin. Neurobiol. 1997; 7: 310-315Crossref PubMed Scopus (332) Google Scholar). Interestingly, it has recently been found that some of these proteins are also expressed in glial cells (30Parpura V. Fang Y. Basarsky T. Jahn R. Haydon P.G. FEBS Lett. 1995; 377: 489-492Crossref PubMed Scopus (169) Google Scholar, 31Jeftinija S.D. Jeftinija K.V. Stefanovic G. Brain Res. 1997; 750: 41-47Crossref PubMed Scopus (93) Google Scholar). These findings, in addition to recent data demonstrating that bradykinin and prostaglandins stimulate a Ca2+-dependent release of glutamate from astrocytes (32Parpura V. Basarsky T.A. Liu F. Jeftinija K. Jeftinija S. Haydon P.G. Nature. 1994; 369: 744-747Crossref PubMed Scopus (1401) Google Scholar, 33Bezzi P. Carmignoto G. Pasti L. Vesce S. Rossi D. Rizzini B.L. Pozzan T. Volterra A. Nature. 1998; 391: 281-285Crossref PubMed Scopus (989) Google Scholar), suggest the presence of a regulated secretory mechanism in at least certain types of glial cells. However, the presence of classical synaptic-like vesicles or dense-core granules in glial cells has never been reported. To study whether astrocytes contain regulated secretory vesicles, we endeavored to determine the intracellular localization and the secretory pathway taken by SgII in cultured hippocampal astrocytes. In this study, dense-core vesicles containing SgII were identified for the first time in astrocytes. Moreover, our results demonstrated that SgII release could be stimulated by different secretagogues including bradykinin, cyclic AMP, ionomycin, and phorbol 12-myristate 13-acetate (PMA). In Ca2+-containing media the ionophore in combination with PMA appeared to be the most effective stimulus for SgII release. Furthermore, treatments with ionomycin or ionomycin plus PMA were found to produce large [Ca2+]i increases in hippocampal astrocytes. The antibodies against rat SgII and chromogranin B (CgB) were raised in rabbits, purified by affinity chromatography, and characterized as described previously (34Rosa P. Hille A. Lee R.W. Zanini A. De Camilli P. Huttner W.B. J. Cell Biol. 1985; 101: 1999-2011Crossref PubMed Scopus (306) Google Scholar). New antisera against the granins were prepared starting from rat pheochromocytoma cells (PC12). Briefly, the heat-stable protein fraction of PC12 cells, enriched in SgII and CgB, was subjected to DEAE-cellulose chromatography and preparative two-dimensional polyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose filters. The filters were stained with Ponceau S (Serva Finebiochemica, Heidelberg, Germany), and the pieces containing SgII and CgB were excised, crushed to a fine powder using a glass potter in liquid N2, and resuspended in phosphate-buffered saline, pH 7.4. The filter homogenates were emulsified with complete Freund's adjuvant for the first injection and incomplete Freund's adjuvant for the booster injections. For each injection, 100 or 50 μg of purified protein were used. The anti-SgII and -CgB antibodies were purified by affinity chromatography as described previously (22Rosa P. Weiss U. Pepperkok R. Ansorge W. Niehrs C. Stelzer E.H. Huttner W.B. J. Cell Biol. 1989; 109: 17-34Crossref PubMed Scopus (82) Google Scholar). The specificity of the antibodies was tested by immunoblotting, immunoprecipitation, and immunofluorescence. The monoclonal antibodies against tubulin, glial fibrillary acidic protein (GFAP), microtubule-associated protein-2, anti-synaptophysin, and anti-syntaxin 1A (HPC-1) were obtained from Roche Molecular Biochemicals. The antisera against galanin, Met-enkephalin, substance P, and cholecystokinin 26–33 were purchased from Peninsula Laboratories Inc. (Belmont, CA). The antibodies against nestin, mannosidase II, and the α-subunit of Na+/K+-ATPase were kind gifts of Dr. W. B. Huttner (University of Heidelberg, Germany) and Dr. M. G. Farquhar (University of California at San Diego, La Jolla, CA) and G. Pietrini (University of Milan), respectively. Secondary antibodies conjugated to fluorescein isothiocyanate, Texas Red, 10- or 5-nm gold particles, peroxidase, and protein A conjugated to 5-nm gold particles were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The PC12 cells were grown as described previously (35Rosa P. Mantovani S. Rosboch R. Huttner W.B. J. Biol. Chem. 1992; 267: 12227-12232Abstract Full Text PDF PubMed Google Scholar). Primary astrocyte cultures were prepared from embryonic day 18 (E18) or post-natal (day 2) rat hippocampi, cerebral cortex, and cerebellum as described by McCarthy and De Vellis (36McCarthy K.D. De Vellis J. J. Cell Biol. 1980; 85: 890-902Crossref PubMed Scopus (3366) Google Scholar) and Banker and Goslin (37Banker G.A. Goslin K. Culturing Nerve Cells. MIT Press, Cambridge, MA1991Google Scholar). After dissection, tissues were incubated for 15 min at 37 °C in 0.25% trypsin and then gently dissociated by trituration with a fire-polished Pasteur pipette. The cells were plated onto glass coverslips (Zeus Super, Italy) or Petri dishes (Falcon, Becton Dickinson, Meylan Cedex, France) at a density of 0.5 × 106 cells/ml. The cultures were grown in minimal essential medium supplemented with 20% fetal bovine serum (Life Technologies, Inc.) and glucose at a final concentration of 5.5 g/liter. To compare the relative density of SgII-containing vesicles from astrocytes and PC12 cells, homogenates prepared from both types of cells were separated on sucrose equilibrium gradients as described (38Navone F. Di Gioia G. Jahn R. Browning P. Greengard P. De Camilli P. J. Cell Biol. 1989; 109: 3425-3433Crossref PubMed Scopus (80) Google Scholar) with the following modifications. PC12 cells and astrocytes, grown on Petri dishes until near confluence, were scraped from the dishes, washed, and resuspended 1:4 in homogenization buffer (10 mm Hepes-KOH, pH 7.4, 250 mm sucrose, 1 mm magnesium acetate, 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml pepstatin, 10 μg/ml aprotinin). The cells were homogenized using a cell cracker (European Molecular Biology Laboratory, Heidelberg, Germany) and centrifuged at 1,000 × g for 10 min to prepare the post-nuclear supernatants (PNS). The PNS was loaded onto a 0.4–1.8m sucrose gradient and spun in a 41 SW rotor (Beckman Instruments) at 25,000 rpm for 18 h. Fractions (1 ml) were collected and analyzed by SDS-PAGE followed by Western blotting as described (22Rosa P. Weiss U. Pepperkok R. Ansorge W. Niehrs C. Stelzer E.H. Huttner W.B. J. Cell Biol. 1989; 109: 17-34Crossref PubMed Scopus (82) Google Scholar, 39Passafaro M. Rosa P. Sala C. Clementi F. Sher E. J. Biol. Chem. 1996; 271: 30096-30104Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Briefly, after electrophoresis proteins were transferred onto nitrocellulose filters. After incubation in blocking buffer (5% milk, 25 mm Tris-HCl, pH 7.5, 150 mm NaCl), the filters were then labeled with primary antibodies followed by the appropriate secondary antibodies conjugated to peroxidase diluted in blocking buffer containing 0.3% Tween 20. After extensive washing, the immunodecoration pattern was revealed using an enhanced chemiluminescence system (SuperSignal from Pierce) following the manufacture' s protocol. To clarify SgII metabolism in astrocytes, different procedures for protein radiolabeling were performed. For pulse-chase experiments, 18-day-cultured astrocytes were preincubated for 4 h in cysteine/methionine-free DMEM. Cells were then labeled with 300 μCi/ml Pro-mix (SJQ, Amersham Pharmacia Biotech) for 60 min in the same medium. After pulse, cells from one dish were washed at 0 °C with ice-cold phosphate-buffered saline and then solubilized in 1% Triton X-100, 50 mm Tris-HCl, pH 7.4, 10 mm EDTA, 500 mm NaCl, and protease inhibitors (solubilization buffer). Labeled cells in parallel dishes were chased in DMEM supplemented with 2 times concentrated methionine and cysteine for 2 consecutive periods of 60 and 180 min. The media were then collected and centrifuged at 13,000 rpm for 10 min. The remaining cells were solubilized as described before. SgII was immunoprecipitated from aliquots of the clear cell lysates (150 μg of proteins) and from corresponding aliquots of the medium samples, as described above. For long-labeling and stimulation experiments, astrocytes were preincubated in sulfate-free DMEM for 2 h and then incubated overnight in medium containing 500 μCi/ml [35S]sulfate (SJS1, Amersham Pharmacia Biotech). After labeling, the cells were rinsed in chase medium (standard DMEM supplemented with 1.6 mm Na2SO4 and 1% fetal bovine serum) and then incubated for 4 h in the same medium. To analyze regulated secretion, the cells were subsequently incubated in DMEM supplemented with 50 mm KCl, 1 μm bradykinin, 100 nm PMA, 5 mmdibutyryl-cAMP, 1 μm ionomycin, or 1 μmionomycin plus 100 nm PMA (Sigma) or in standard DMEM. The various media were collected and centrifuged as described above. The cells were washed and solubilized in ice-cold solubilization buffer containing 0.3% Tween 20 instead of Triton X-100. For immunoprecipitation, the cell lysates were spun for 5 min at 13,000 rpm. The protein concentrations were determined using a Bio-Rad protein assay. The cell lysates (10 μg) and aliquots of their respective media were then analyzed by SDS-PAGE followed by fluorography as described previously (35Rosa P. Mantovani S. Rosboch R. Huttner W.B. J. Biol. Chem. 1992; 267: 12227-12232Abstract Full Text PDF PubMed Google Scholar). SgII was immunoprecipitated from the clear cell lysates containing equal amounts of proteins (70 μg) and equivalent aliquots of the corresponding media (see above). Aliquots of the media and clear cell lysates were diluted in immunoprecipitation buffer at a final concentration of 50 mm Tris-HCl, pH 7.5, 10 mm EDTA, 500 mm NaCl, 0.5% (or 1%) Triton X-100, and 1 mm phenylmethylsulfonyl fluoride and incubated overnight at 4 °C with anti-rat SgII or anti-rat CgB antiserum. The amounts of antisera were sufficient to bind all of the granins present in the samples (34Rosa P. Hille A. Lee R.W. Zanini A. De Camilli P. Huttner W.B. J. Cell Biol. 1985; 101: 1999-2011Crossref PubMed Scopus (306) Google Scholar). The immunocomplexes were collected by binding to protein A conjugated to Sepharose CL-4B (Amersham Pharmacia Biotech). After extensive washing, the immunoprecipitates were analyzed by SDS-PAGE followed by fluorography. Quantitation of labeled SgII in cell lysates or media after immunoprecipitation was carried out using different methods. Fluorograms were quantitated directly by densitometric scanning using an LKB Ultrascan (Amersham Pharmacia Biotech), or fluorogram images were acquired by means of a ARCUS II scanner (Agfa-Gevaert N.V., Mortsel, Germany), and the density of the bands was quantitated using the Image program (National Technical Information Service, Springfield, VA), version 1.55. For each pharmacological treatment, results obtained from three independent experiments were expressed as a percentage of the controls and analyzed using the statistical program StatWorks (Cricket Software Inc., Philadelphia, PA). After long-labeling, the radioactivity incorporated in the SgII bands was quantitated by liquid scintillation counting. The bands corresponding to [35S]sulfate-labeled SgII were excised from the gels and dissolved by overnight treatment with 35% hydrogen peroxide at 80 °C. After dilution in scintillation liquid (Ultima Gold, Packard Instrument Co.), radioactivity was counted using a liquid scintillation analyzer. The data are presented as mean ± S.D. For double immunofluorescence, the cells were incubated in 120 mm phosphate buffer, pH 7.2, containing 4% formaldehyde and 4% sucrose. After fixation, cells were immunostained using primary antibodies followed by the appropriate secondary antibodies, as described previously (22Rosa P. Weiss U. Pepperkok R. Ansorge W. Niehrs C. Stelzer E.H. Huttner W.B. J. Cell Biol. 1989; 109: 17-34Crossref PubMed Scopus (82) Google Scholar). In some experiments, the cells were treated with 10 μg/ml cycloheximide (Sigma) before fixation. Specificity of SgII immunostaining was demonstrated by antisera preadsorption with the purified antigen. Briefly, 3 μg of affinity-purified anti-SgII-antibody were preadsorbed with 15 μg of SgII purified by two-dimensional PAGE and transferred onto nitrocellulose filters. The filter pieces containing the desired amount of SgII were preincubated in 20 mmphosphate buffer, pH 7.3, 500 mm NaCl, 0.2% gelatin (immunofluorescence buffer) for 3 h at room temperature and then incubated with affinity-purified anti-SgII antibodies for 18 h at 4 °C. Electron or immunoelectron microscopy was performed as described previously (20Rosa P. Bassetti M. Weiss U. Huttner W.B. J. Histochem. Cytochem. 1992; 40: 523-533Crossref PubMed Scopus (31) Google Scholar). After fixation, the astrocytes were scraped from the dishes and pelleted in an Eppendorf centrifuge. The pellets were either dehydrated and infiltrated in Epon 812 for preparation of plastic sections or infiltrated in 2.1 m sucrose for the preparation of cryosections. The ultra-thin frozen sections were immunolabeled using anti-rat SgII antibodies revealed with protein A conjugated to 5-nm gold particles. Double labeling with anti-SgII and anti-GFAP was revealed using anti-rabbit IgG conjugated to 10-nm gold particles and anti-mouse IgG antibodies conjugated to 5-nm gold particles. Astrocytes were loaded for 1 h at 37 °C with 3–4 μm Fura-2 pentacetoxymethyl ester in Krebs-Ringer solution buffered with Hepes (150 mm NaCl, 5 mm KCl, 1.2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, 10 mm Hepes/NaOH, pH 7.4), washed in the same solution to allow deesterification of the dye, and transferred to the recording chamber of an inverted microscope (100 TV; Zeiss) equipped with a calcium imaging unit. For the assays, a modified CAM-230 dual wavelength microfluorometer (Jasco, Tokyo, Japan) was used as a light source. Fluorescence images were collected with an intensified CCD camera (Hamamatsu Photonics), digitized, and integrated in real time in an image processor developed in the laboratory (40Grohovaz F. Zacchetti D. Clementi E. Lorenzon P. Meldolesi J. Fumagalli G. J. Cell Biol. 1991; 113: 1341-1350Crossref PubMed Scopus (46) Google Scholar). Image files were processed off-line to convert fluorescence data into [Ca2+]i maps according to the 340/380-nm excitation wavelength ratio method (41Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Mean ratio values in discrete areas of interest were calculated from sequences of images to obtain quantitative temporal analyses. To investigate the possible presence of regulated secretory vesicles in astrocytes, we decided to investigate the intracellular distribution of SgII, a diffuse marker of dense-core granules (17Huttner W.B. Gerdes H.H. Rosa P. Trends Biochem. Sci. 1991; 16: 27-30Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 18Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (609) Google Scholar, 19Rosa P. Gerdes H.H. J. Endocrinol. Invest. 1994; 17: 207-225Crossref PubMed Scopus (105) Google Scholar), in glia primary cultures. The cell cultures were prepared from brains isolated from 18-day-old embryos or 2–5-day post-natal rats as described (37Banker G.A. Goslin K. Culturing Nerve Cells. MIT Press, Cambridge, MA1991Google Scholar). After dissection, the cells were maintained in culture in the presence of fetal bovine serum without supplements to promote survival and proliferation of astrocytes. After 3 weeks, the majority of the cells showed a polygonal shape and expressed high levels of the intermediate filament protein GFAP, which is usually expressed by differentiated astrocytes (42Eng L.F. J. Neuroimmunol. 1985; 8: 203-214Abstract Full Text PDF PubMed Scopus (601) Google Scholar) (Figs.Figure 1, Figure 2, Figure 3, Figure 4and data not shown). Cells expressing markers of the neuronal phenotype (i.e. neurofilament proteins and microtubule-associated protein-2) were not detected (data not shown). In 3-week old cultures prepared from embryonic hippocampi, only a small amount of the cells (10%, n = 2) were found to synthesize the intermediate filament protein marker of neuronal stem cells, nestin (43Frederiksen K. McKay R.D. J. Neurosci. 1988; 8: 1144-1151Crossref PubMed Google Scholar), thus suggesting that the majority of the astrocytes present in the primary culture prepared were largely differentiated.Figure 3Double-immunofluorescence showing the intracellular storage of SgII in astrocytes. Three-week-old astrocyte cultures were incubated for 4 (A–A′) and 18 (B–B′) h with 10 μg/ml cycloheximide. After drug treatment, the cells were fixed and immunolabeled for GFAP (A and B) and SgII (A′ andB′). In the cycloheximide-treated cells, SgII immunoreactivity is only observed in dot-like structures (A′ and B′, arrows), which remain for a long time, whereas the Golgi-like staining is completely abolished.Bars, 10 μm.View Large Image Figure ViewerDownload (PPT)Figure 2Expression of SgII in hippocampal astrocytes in 18-h-old culture. The astrocytes were prepared using E18 rat hippocampi. After 18 h of culture, the cells were fixed and then stained with anti-GFAP antibodies (A and B) and affinity-purified antibodies against SgII (A′ andB′). The arrows indicate the astrocytes showing immunoreactivity for SgII. Bars, 10 μm.View Large Image Figure ViewerDownload (PPT)Figure 1Immunolocalization of SgII in astrocytes. Astrocytes were prepared from post-natal (A, A′) or E18 (B–D) rat hippocampi as described under “Experimental Procedures.” After 3 weeks in culture, the cells were fixed and double-immunolabeled using monoclonal antibodies against GFAP (diluted 1:200 (A and C)) or tubulin (diluted 1:100 (B)) and affinity-purified polyclonal anti-SgII antibody (1.5 μg/ml (A′–C′)). InD, the cells were single-labeled with a polyclonal antibody against mannosidase II, a marker of the Golgi cisternae. Note the immunoreactivity for SgII in the Golgi apparatus (arrowheadsin A′–C′ compared with D) and in dot-like structures dispersed in the cytoplasm (arrows).Bars, 10 μm.View Large Image Figure ViewerDownload (PPT) The 3-week-old astrocyte cultures prepared from post-natal or E18 rats were then p" @default.
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• W1992807239 date "1999-08-01" @default.
• W1992807239 modified "2023-10-12" @default.
• W1992807239 title "A Regulated Secretory Pathway in Cultured Hippocampal Astrocytes" @default.
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