Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), andbiliverdin. We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression byWestern blot in primary astroglial cells during differentiation and after exposure to glutamate (100 lM). CLSM analysis of immunostainedHO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and adecrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels. The distribution of HO-1 protein undergoesmodification in the various cellular compartments. Furthermore, localization of the protein in untreated astrocytes at 7 daysappeared prevalently localized in the cytosol and in the perinuclear region. In contrast, at 14 and 21 days, fluorescence detectionsuggests that HO-1 was present also in the nucleus, and in the nucleoli. Fluorescence intensity significantly increased in glutamatetreatedastrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli. Theinvolvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cellswith GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors. Westernblot analysis of HO-1 confirmed the CLSM results. Our results demonstrate that changes in HO-1 protein expression and localizationin primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases.