No abstract

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K+ depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E2 treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells’ population growth by inducing maturation and differentiation.

Cultured rat cortical astrocytes, in addition to a variety of voltage-sensitive potassium channels, also express anion channels. However, the behavior and regulation of these anion channels have been far less studied. This paper describes a patch-clamp study on a voltage-sensitive 200-300 pS high-conductance single-channel anion current, which seems to possess at least five different open sublevels or, alternatively, be formed from five or more small-conductance ion channels linked together. This channel is voltage dependent, showing a bell-shaped open probability curve with highest open probability close to the reversal potential (zero-current). Although potassium channels are commonly detected in astrocytes in cell-attached and excised patches with both normal osmolarity and hypoosmotic solutions, the occurrence of the anion channel is clearly increased in isolated patches when hypoosmotic bath solutions are used. Also, cell aging in culture and the preparation of secondary cell cultures by trypsinization seem to increase the rate of occurrence of the anion channel. Though this channel is more routinely seen when a membrane patch is excised from the cell, occasionally cell-attached configurations with instant channel activity can be formed. While the modulation of this anion channel was being studied, it was found to be blocked by an anion transport inhibitor, L-644,711, reported to affect cell volume regulation in astrocytes.

Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.

Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3-10-dayold rats frequently showed increased intracellular Ca 2c oncentration responses to L-glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonm [5-hydroxytryptamine(5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca2~responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of, or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAPnegative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca2r esponses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.