Intracellular calcium and survival of tadpole forebrain cells in anoxia.
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 BTC) 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% N2) and measurements of [Ca2+]i and cell survival made from 1 h to 18 h. Intracellular [Ca2+] increased significantly after 3-6 h anoxia (P<0.05), regardless of the type of Ca2+ indicator used; however, there were substantial differences in the measurements of [Ca2+]i 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 micromol l(-1) after 4-6 h 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 reoxygenated for 12 h to 16 h, 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.
Hedrick
Michael S
MS
Department of Anesthesia, University of California, San Francisco, CA 94143-0542, USA. mhedrick@csuhayward.edu
Fahlman
Christian S
CS
Bickler
Philip E
PE
D000704:Analysis of Variance; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D005453:Fluorescence; D000860:Hypoxia / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology; D007814:Larva / Q000378:metabolism / Q000502:physiology; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D011419:Propidium; D016548:Prosencephalon / Q000166:cytology / Q000378:metabolism*; D011892:Rana catesbeiana / Q000378:metabolism* / Q000502:physiology
D011419:Propidium; D002118:Calcium