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Department of Physiology, University College London.
1. Impaired intracellular Ca2+ concentration ([Ca2+]i) regulation may underlie alterations in neuronal function during hypoxia or hypoglycaemia and may initiate cell damage. We have used the Ca2(+)-sensitive fluorophore, Fura-2, to study the regulation of [Ca2+]i in neurones isolated from mouse dorsal root ganglia. Mean resting [Ca2+]i was 163 +/- 11 nM (mean +/- S.E.M., n = 38). 2. Depolarization by exposure to 20 or 30 mM-K+ caused a rapid Co2(+)- and Cd2(+)-sensitive rise in [Ca2+]i, which subsequently declined with a time course usually fitted by the sum of two exponential functions. 3. Interference with mitochondrial function (by CN- or FCPP) or with glycolysis (by glucose removal) all raised [Ca2+]i by up to 220%. Addition of FCCP in the presence of CN- further increased [Ca2+]i. The response to CN- was still seen in the absence of extracellular Ca2+, although it attenuated rapidly, indicating release from an intracellular store. 4. Either CN- or glucose removal increased the rise in [Ca2+]i induced by K+ 2- to 3-fold and slowed recovery, suggesting interference with sequestration or extrusion of [Ca2+]i. 5. Resting [Ca2+]i rose when external Na+ was replaced by Li+ or N-methyl-D-glucamine, demonstrating the presence of a Na(+)-Ca2+ exchange process. However, Na+ replacement had only a slight effect on the handling of a Ca2+ load. 6. We conclude that (i) Ca2+ is released into the cytoplasm from intracellular organelles when energy supplies are reduced: (ii) that the extrusion or sequestration of Ca2+ entering the cell during electrical activity is rapidly impaired by interference with mitochondrial metabolism: and (iii) Na(+)-Ca2+ exchange makes only a small contribution to intracellular Ca2+ homeostasis. 7. [Ca2+]i would thus be expected to rise in vivo during hypoxia or hypoglycaemia and may initiate alterations in neuronal function. However, if a rise in Ca2+ is an important cause of cell damage in cerebral hypoxaemia, the combination of excitation and hypoxia will lead to the largest increases in [Ca2+]i, while hypoxia alone appears to cause only a small increase in [Ca2+]i in quiescent cells.
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