CELLULAR-ENERGY METABOLISM, TRANS-PLASMA AND TRANS-MITOCHONDRIAL MEMBRANE-POTENTIALS, AND PH GRADIENTS IN MOUSE NEURO-BLASTOMA

A method for quantitative evaluation of transmembrane electrical potential and pH gradients across a subcellular compartment in an intact cell is presented. This approach has been applied in studies of mouse neuroblastoma C-1300 clone NB41A3, in which the transmembrane electrical potential and pH gradients and the mitochondrial volume percent have been determined. Membrane potentials and pH gradients were measured by two different methods. Equilibrium distributions of [3H]triphenylmethyl phosphonium and [14C]thiocyanate ions gave calculated apparent membrane potentials of -77.0 and -29.6 mV, respectively, at 20-25°C; a value of -60.8 mV was obtained from microelectrode measurements. Equilibrium distributions of weak acids ([14C]trimethylacetic acid and 5,5-di[14C]methyl-2,4-oxazolidine-dione) and of weak bases ([14C]dimethylamine and [14C]trimethylamine) gave calculated upper and lower limits of the pH gradient (Δ pH = pH(e) - pH(i)) of -0.14 and -0.21 pH unit, respectively. The micro-electrode measurements showed that the intracellular pH is within 0.1 of a pH unit or less of the extracellular pH over the extracellular pH range of 7.35-6.85. The mitochondrial volume percent calculated on the basis of the measured cytochrome c content is 5.6 ± 1.2% and compares well with estimates of 5.4 ± 1.1% obtained from 25 electron micrographs. Measurements of the cellular energetic parameters gave values within the range found in other cells and perfused organs. Comparison of the results of the microelectrode and equilibrium measurements permits estimates of the electrical potential and pH gradients across the mitochondrial membrane (mitochondria-to-cytoplasm gradients) to be made and suggests that the trans-mitochondrial membrane protonmotive force in the intact cell cannot be greater than -143mV.