Measurement of the intramitochondrial volume in hepatocytes without cell disruption and its elevation by hormones and valinomycin

Abstract

Methods have been developed to measure the lysophospholipid content and matrix volume of liver cell mitochondria in situ in order to test the hypothesis that these parameters may be important in the hormonal control of mitochondrial function [Armston, Halestrap & Scott (1982) Biochim. Biophys. Acta 681, 429-439]. No change in the labelling of mitochondrial lysophospholipids with [32P]Pi was detected after treatment of liver cells with glucagon, phenylephrine or vasopressin. Incorporation of [32P]Pi into mitochondrial phosphatidylinositol was enhanced by phenylephrine and vasopressin. Mitochondrial volumes were measured using rapid disruption of cells by sonication into 3H2O and [14C]sucrose or without cell disruption using 3H2O and [14C]mannitol. In control cells the two methods gave values of 1.09 and 0.40 microliters/mg of mitochondrial protein respectively, which represent 19 and 7% respectively of the total cell volume measured with 3H2O and inulin [14C]carboxylic acid. Both methods showed that glucagon, phenylephrine and 1 nm-valinomycin produced significant increases (13% and 26% using sucrose and mannitol respectively) in mitochondrial volume. The increase was coincident with the stimulation of gluconeogenesis from L-lactate and pyruvate and of mitochondrial respiratory chain activity. The effects of glucagon and phenylephrine were additive on both mitochondrial volume and respiratory chain activity, but not on gluconeogenesis. Liver cells exposed to gluconeogenic hormones or low concentrations of valinomycin showed a decrease in light scattering at 520 nM correlating with the change in mitochondrial volume but without a change in whole-cell volume. The time course and hormone sensitivity of this response were similar to those for the hormonal stimulation of gluconeogenesis. The light-scattering response to glucagon, phenylephrine and vasopressin, but not to valinomycin, were greatly reduced or abolished in Ca2+-free media.