Intracellular calcium and survival of tadpole forebrain cells in anoxia

摘要

The frog brain survives hypoxia with a slow loss of energy charge and ion homeostasis. Because hypoxic death in most neurons is associated with increases in intracellular calcium ([Ca2+](i)), we examined the relationship between [Ca2+](i) and survival of a mixed population of isolated cells from the forebrain of North American bullfrog Rana catesbeiana tadpoles. Forebrain cells from stage V-XV tadpoles were isolated by enzymatic digestion and loaded with one of three different calcium indicators (Fura-2, Fura 2-FF and BTQ to provide estimates of [Ca2+](i) accurate at low and high [Ca2+](i). Propidium iodide (PI) fluorescence was used as an indicator of cell viability. Cells were exposed to anoxia (100% N-2) and measurements of [Ca2+](i) and cell survival made from 1 h to 18 h. Intracellular [Ca2+](i) increased significantly after 3-6h anoxia (P < 0.05), regardless of the type of Ca2+ indicator used; however, there were substantial differences in the measurements of [Ca2+], with the different indicators, reflecting their varying affinities for Ca2+. Resting [Ca2+](i) was approximately 50 nmol l(-1) and increased to about 9-30 mu mol l(-1) after 4-6h anoxia. The significant increase in [Ca2+](i) during anoxia was not associated with significant increases in cell death, with 85-95% survival over this time period. Cells exposed to anoxia for 18 h, or those made anoxic for 4-6 and re-oxygenated for 12h to 16h, had survival rates greater than 70%, but survival was significantly less than normoxic controls. These results indicate that large increases in [Ca2+](i) are not necessarily associated with hypoxic cell death in vertebrate brain cells.