Effects of metabolic blockade on the regulation of intracellular calcium in dissociated mouse sensory neurones

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). 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. 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. 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). 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. 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. [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.