title: Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate.
abstract: Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), and biliverdin. We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression by Western blot in primary astroglial cells during differentiation and after exposure to glutamate (100microM). CLSM analysis of immunostained HO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and a decrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels. The distribution of HO-1 protein undergoes modification in the various cellular compartments. Furthermore, localization of the protein in untreated astrocytes at 7 days appeared prevalently localized in the cytosol and in the perinuclear region. In contrast, at 14 and 21 days, fluorescence detection suggests that HO-1 was present also in the nucleus, and in the nucleoli. Fluorescence intensity significantly increased in glutamate-treated astrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli. The involvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cells with GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors. Western blot analysis of HO-1 confirmed the CLSM results. Our results demonstrate that changes in HO-1 protein expression and localization in primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases.
authors.affiliation: Department of Biochemical, Physiological and Nutritional Sciences, University of Messina, Italy. livolti@tiscalinet.it
authors.forename: Giovanni
authors.initials: G
authors.lastname: Li Volti
authors.forename: Riccardo
authors.initials: R
authors.lastname: Ientile
authors.forename: Nader G
authors.initials: NG
authors.lastname: Abraham
authors.forename: Angelo
authors.initials: A
authors.lastname: Vanella
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cannavò
authors.forename: Francesco
authors.initials: F
authors.lastname: Mazza
authors.forename: Monica
authors.initials: M
authors.lastname: Currò
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Raciti
authors.forename: Roberto
authors.initials: R
authors.lastname: Avola
authors.forename: Agata
authors.initials: A
authors.lastname: Campisi
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000201:enzymology* / Q000378:metabolism; D001569:Benzodiazepines / Q000494:pharmacology; D015153:Blotting, Western; D002454:Cell Differentiation; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D003720:Densitometry; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology*; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis*; D051547:Heme Oxygenase-1; D015151:Immunoblotting; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000378:metabolism; D013997:Time Factors
chemical_list: D018691:Excitatory Amino Acid Antagonists; D005971:Glutamates; D018091:Receptors, AMPA; C061950:GYKI 52466; D001569:Benzodiazepines; D018698:Glutamic Acid; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D005974:Glutamate-Ammonia Ligase