Mechanisms underlying calcium homeostasis in isolated hepatocytes.

The steady state relationship between intra- and extramitochondrial free Ca2+ across the inner mitochondrial membrane has been investigated in isolated liver mitochondria. The extramitochondrial free Ca2+ concentration was essentially independent of the mitochondrial calcium content above 4 nmol/mg of protein. Below this value, a decrease in the mitochondrial calcium content was accompanied by a decrease in the extramitochondrial free Ca2+ concentration. The experimental data are compatible with a model in which the steady state distribution of calcium is described in terms of the kinetic parameters of the separate carriers catalyzing Ca2+ influx and efflux across the mitochondrial inner membrane. The corresponding relationship between cytosolic free Ca2+ concentration and the amounts of calcium in the mitochondria and endoplasmic reticulum was investigated in isolated river cells over a range of cellular Ca2+ contents by using a nondisruptive technique based on the selective release of calcium from mitochondrial and total cellular pools by addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and A23187, respectively. A net increase in cell calcium from 1 to 5 nmol/mg dry weight, increased the cytosolic free Ca2+ concentration from 0.1 to about 0.3 microM and increased the calcium contents of both mitochondria and endoplasmic reticulum. Above 5 nmol of calcium/mg cell dry weight, the endoplasmic reticulum calcium pool became filled, and further increases in calcium content were accounted for by increases of the mitochondrial pool but no further increase of the cytosolic free Ca2+ concentration. These studies and experiments with mixtures of isolated microsomes and mitochondria suggest that, in cells as normally isolated (containing 5 to 6 nmol of calcium/mg dry weight), the endoplasmic reticulum is saturated with calcium and is unlikely to play a major role as an intracellular calcium buffer. The in situ mitochondrial calcium content is sufficiently high (approximately 16 nmol/mg of protein) for these organelles to buffer effectively the cytosolic free Ca2+ concentration at a value of about 0.3 microM. In addition, it may be concluded that intramitochondrial Ca2+-dependent enzymes will be exposed to saturating concentrations of free Ca2+.