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.

Control of respiration and oxidative phosphorylation in isolated rat liver cells

“…One method of following changes of matrix volume in liver cells is via light scattering, an increase in light scattering indicating a decrease in matrix volume [29]. We found that vasopressin addition (2.5-75 nM) resulted in a small stable increase in light scattering (increased absorbance at both 520 nm and 540nm) in both fed and starved cells, suggesting that the stimulation in respiration could not be accounted for by an increase in matrix volume.…”

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

“…We first tested the applicability of this assay to mitochondria in situ with permeabilized hepatocytes (Fig. 6B), in which mitochondrial swelling can be monitored by cell suspension absorbance (79,80) and correlated with the DTNB reduction rate as well as mitochondrial functional parameters. Hepatocytes were permeabilized with saponin according to the protocol applied to ␤-cells and loaded with Ca 2ϩ in the presence of P i .…”

Mitochondrial membrane permeabilization produced by PTP, Bax and apoptosis: a 1H-NMR relaxation study

“…23 In brief, mitochondria were incubated (500 mg protein.ml À1 ) at room temperature in 250 mM sucrose, 80 mM KCl, K-malate 10 mM, K-succinate 8 mM, ATP-Na 2 1 mM, EDTA-Na 4 1 mM, MOPS-K 20 mM (pH 7.4) in the absence of MgCl 2 . PTP opening was induced by raising the free Ca 2 þ concentration with known volumes of CaCl 2 stock solution calculated by using MaxChelator software (http:// www.stanford.edu/~cpatton/maxc.html).…”