Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (86Rb+) influx suppressed by 10 microM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl- cotransport. Inhibitors of protein kinase C (10 microM polymyxin B and l microM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70-80%. Similar results were obtained with calmodulin antagonist R24571 (10 microM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl- cotransport was also suppressed by protein kinase C activator PMA (l microM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 microM vanadate, 5 mM fluoride and 0.5 microM okadaic acid) activated greatly the bumetanide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl- cotransport in glial cells.

Primary human glial fibrillary acidic protein-positive (GFAP+) brain cells (enriched population) have successfully been infected with human immunodeficiency virus type 1 (HIV-1) in vitro, when cocultivated with HIV-1-producing H9 cells. Direct incubation of brain cells with HIV-1 resulted only in a limited infection. The percentage of HIV+ cells increased from 5% in passage 1 to 40% in passage 8. Simultaneously with the increase of infected cells, the reverse transcriptase activity in the culture medium increased and reached maximal values in passage 8. The infected cells also produced intact viral particles. In the early phase of cultivation the HIV-infected cells displayed a significantly higher proliferation rate than the uninfected controls. At passage number 8 the HIV-infected GFAP+ cells had almost totally lost the ability to grow, while the controls proliferated at a rate almost unimpaired from the beginning of the cultivation. Up to 10 to 15% of the HIV-infected GFAP+ cells contained at passage number 5 more than 3 nuclei. Memantine (1-amino-3,5-dimethyladamantane), a blocker of the N-methyl-D-aspartate receptor channels, was found to display a significant anti-HIV effect (at a concentration of 1 microgram/ml) on enriched cultures of GFAP+ cells in vitro.

2-Hexadecenal (2HD) formation in the organism occurs via irreversible enzymatic degradation of sphingosine-1-phosphate or nonenzymatic γ-, UV-, or HOCl-induced destruction of a number of sphingolipids including S1P. The current research focuses on the study of 2HD effects on C6 glioma cells growth. The results obtained show that 2HD causes a dose-dependent decrease in proliferative and mitotic indices.The change in the mitotic index is due to the redistribution of cells in the different phases of mitosis. These processes are accompanied by cytoskeleton rearrangement and changes in cell morphology, which are expressed in F-actin redistribution, change in the number and type of filopodia and fibrils, leading to cell shape changes, decrease in intercellular contacts and monolayer rarefaction. Cells treatment with 2HD leads to apoptosis induction and signalling pathways modification, including activation of JNK, p38, and ERK1/2 MAPK but not PI3K. The effects observed are not related to the cytotoxicity of 2HD. Significance of the study: 2HD-an unsaturated aldehyde, which level can rise under conditions of oxidative stress as a result of nonenzymatic sphingolipids' destruction. The mechanisms of 2HD action on various cell types have not been sufficiently studied. Therefore, the study on functional role of this aldehyde in different cell types that may be its target is relevant. This study demonstrated that 2HD inhibits growth of C6 glioma cells due to modification of intracellular processes of signal transduction, cytoskeleton rearrangement, change in the mitotic regimen and apoptosis induction.

No abstract

We studied the effect of a donor of peroxynitrite, SIN-1, on the morphological characteristics of interweaved rat С6 glioma cells, on menadione-induced production of superoxide anion radicals, and on the concentration of Са 2+ in these cells. In concentrations of 1.25·10 -4 to 2.5·10 -7 М, SIN-1 demonstrated cytotoxic and antimitogenic effects. This donor of peroxynitrite caused abnormal modifications of the size of С6 cells and the structure of cellular organelles, intensified in a dose-dependent manner the release of Са 2+ from cellular stores into the cytoplasm, and suppressed menadione-induced production of superoxide anion radicals. Therefore, it should be believed that peroxynitrite exerts a modifying effect on the processes of mitotic division and induces apoptosis; it is also involved in the processes of intracellular signalling providing an increase in the concentration of cytosolic Са 2+ and a decrease in the redox activity of cells.