Isolation, cloning and characterization of a putative type-1 astrocyte cell line.

We have established a permanent cell line (1H91) of putative type-1 astrocyte precursor cells that were clonally derived from a single cell isolated from E16 mouse cerebellum. Epidermal growth factor (EGF) and transforming growth factor (TGF alpha) are strong mitogens for 1H91 cells (ED50 of 9.02 + 1.74 ng/ml and 15.98 +/- 2.34 ng/ml, respectively), while basic fibroblast growth factor (bFGF) is only weakly mitogenic and platelet derived growth factor (PDGF) has no mitogenic activity. In the proliferative state, the 1H91 cells are immunohistochemically positive for nestin and vimentin, and negative for A2B5, CNPase, neurofilament (NF), and neuron specific enolase (NSE). The majority of EGF-treated 1H91 cells are not immunoreactive for glial acid fibrillary protein (GFAP). In the presence of 5 ng/ml bFGF, 1H91 cells become non-mitotic and develop a morphology consistent with a fibrous astrocyte. In contrast to the proliferating cultures, the bFGF treated cultures were strongly immunoreactive for GFAP, only mildly immunoreactive for nestin and vimentin, and negative for A2B5, CNPase, NF, and NSE. Type-1 astrocytes are known to proliferate in response to EGF, and are immunohistochemically GFAP positive, A2B5 negative, and CNPase negative [38]. However, type-1 astrocytes only develop a fibrous morphology during the process of reactive gliosis [31]. Since EGF is a strong mitogen for 1H91 cells, and these cells may be differentiated into GFAP positive, A2B5 negative, CNPase negative astrocytes, we conclude that 1H91 cells conform to a type-1 astrocyte precursor phenotype. In addition, the fibrous morphology of the bFGF treated 1H91 cells suggests that these cells follow the process of reactive gliosis. Therefore, the 1H91 clonal cell line may provide an in vitro model for studying the underlying cellular mechanisms of the type-1 astrocyte in reactive gliosis.

Differentiation of NG108-15 neuroblastoma cells by serum starvation or dimethyl sulfoxide results in marked differences in angiotensin II receptor subtype expression.

Differentiation of NG108-15 neuroblastoma cells following exposure to either 1.5% dimethyl sulfoxide (DMSO)/0.5% fetal bovine serum (FBS) or serum starvation resulted in significant differences in angiotensin (AT) receptor levels and the AT1/AT2 receptor ratio. When NG108 cells were differentiated for 4 days with DMSO/low serum, the number of AT binding sites increased 30-fold compared with the binding levels on undifferentiated (blast) cells. However, cells differentiated by serum starvation for 4 or 14 days resulted in only a modest 2.5- and fivefold increase in AT receptor levels, respectively, over the levels seen with the undifferentiated cells. KD values for all treatment conditions were not significantly different (0.71 +/- 0.11 nM, p = 0.06). Using the AT1 and AT2 isoform-specific receptor antagonists losartan and PD123319, the relative numbers of AT receptor subtypes on undifferentiated and differentiated cells were determined by competitive inhibition against 125I-[Sar1,Ile8]-angiotensin II (sarile). A majority of the AT receptors on undifferentiated NG108 cells were the AT1 subtype (AT1/AT2 receptor ratio of 8:3). Differentiation by serum starvation and DMSO/low serum treatment resulted in fivefold and 30-fold increases in AT receptor levels, respectively, compared with the levels seen with the undifferentiated cells. Although serum starvation increased the total number of AT1 and AT2 receptors, it did not significantly alter the AT1/AT2 receptor ratio. In contrast, differentiation with DMSO/low serum both increased the total number of AT1 and AT2 receptors and reversed the AT1/AT2 receptor ratio (1:3). The increase in AT receptors following differentiation with DMSO/low serum for 4 days was largely accounted for by an 80-fold increase in the AT2 receptor level. Previous studies by Tallant at al. (1991) and Bryson et al. (1992) reported increased AT2 receptor expression following neuroblastoma differentiation with dibutyryl cyclic AMP and DMSO/low serum, respectively, and suggested a role for the AT2 receptor in neuronal differentiation. In the present study, we have extended these earlier observations by demonstrating that the method of differentiation significantly affects both the AT receptor level and the ratio of AT1 to AT2 receptor expression. Finally, our findings indicate that the AT2 receptor is expressed as a consequence of neuronal maturation and dose not mediate morphological differentiation.