Involvement of two-pore channels in hydrogen peroxide-induced increase in the level of calcium ions in the cytoplasm of human umbilical vein endothelial cells.

Hydrogen peroxide at concentrations below cytotoxic ones causes an increase in the cytoplasmic calcium concentration in human umbilical vein endothelial cells as a result of calcium release from intracellular stores. Two-pore calcium channel blocker trans-NED19 partially suppresses the increase in the level of calcium ions in the cells in response to the addition of hydrogen peroxide. The staining of endothelial cells with the fluorescent stereoisomer cis-NED19 and LysoTracker confirmed the localization of two-pore calcium channels in lysosomes and endolysosomal vesicles.

Suppression of Histamine-Induced Relaxation of Rat Aorta and Calcium Signaling in Endothelial Cells by Two-Pore Channel Blocker trans-NED19 and Hydrogen Peroxide.

The blocker of two-pore channels trans-NED 19 and hydrogen peroxide were found to inhibit histamine-induced relaxation of rat-aorta. The degree of inhibition depended on histamine concentration. The relaxation in response to I µM histamine of rat aorta preconstricted with 30 mM KCI, serotonin, or endothelin- 1, was completely abolished by 30 µM trans-NED 19. On the other hand, trans-NED 19 decreased the relaxation of the aorta in the presence of 10 µM histamine only by 2.1-fold to 2.4-fold, and there was almost no inhibition by trans-NED 19 of the relaxationinduced by 100 ptM histamine.) Relaxation of precontracted with serotonin aorta in response to 10 and 100 µM histamine was reduced by hydrogen peroxide (200 M) by 10- and 2.5-fold, respectively. Suppression of aorta relaxation by trans-NED 19 and H202 correlated with their inhibitory effect on the histamine-induced increase in the cytoplasmic free calcium concentration in human umbilical vein endothelial cells. With the use of a fluorescent probe LysoTracker, the cis-NED19 binding sites were demonstrated to be localized in endolysosomes of the endothelial cells. These data indicate that two-pore calcium channels participate in the histamine-induced endothelium-dependent relaxation of rat aorta. Furthermore, their functional role is exhibited much more clearly at low histamine concentrations. We suggest that hydrogen peroxide evokes depletion of intracellular calcium depots thereby suppressing the response to histamine.

Immunochemical analysis of glial fibrillary acidic protein as a tool to assess astroglial reaction in experimental C6 glioma.

In experiments on Wistar rats with experimental C6 glioma, the immunohistochemical features of the astroglial reaction over 30 days after implantation were characterized. The formation of a glial border consisting of GFAP-positive reactive astrocytes at the periphery of C6 glioma was observed on postimplantation day 3 and until the death of the experimental animals. Reactive astrocytes encompassed not only the primary gliomal focus, but all tumor invasion foci. Quantitative assessment of astroglial reaction around glioma was carried out with immunofluorescent assay of glial fibrillary acidic protein (GFAP) on cerebral sections. The size of glioma and necrotic foci were analyzed morphometrically in parallel with enzyme immunoassay of serum GFAP. A correlation between morphometric indices of glioma and serum level of GFAP was found. It was concluded that serum concentration of GFAP correlated with the size of intracranial glioma, necrotic foci, and most strongly with the degree of reactive astrogliosis. Monitoring of the level of serum GFAP can serve as an additional diagnostic index reporting the state of intracranial glioma.

Characteristics of human neural stem cells in vitro and after transplantation into rat brain.

We studied the effect of culturing conditions on the fate of human neural stem cells after transplantation into rat brain. Human neural stem cells cultured in the presence of mitogens without LIF migrated along the ependyma and cerebral vessels of recipients, but to a great extent degenerated by the 20th day after transplantation. Neural stem cells cultured with LIF migrated, apart from the above mentioned pathways, in the cortex and hippocampus, well survived; proliferating cells were retained 30 days after transplantation.