Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content

Abstract

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.

Control of respiration and ATP synthesis in mammalian mitochondria and cells

“…Addition of calcium or pyrophosphate, or other agents that raise mitochondrial pyrophosphate, was found to increase the volume of isolated liver mitochondria (Davidson & Halestrap, 1987) and heart mitochondria (Halestrap, 1987). The increased mitochondrial volume stimulates the respiratory chain (by an undefined mechanism) at the level of electron flow into the ubiquinone pool, resulting in stimulation of respiration on many different substrates, particularly fatty acids, in liver and heart mitochondria (Davidson & Halestrap, 1987;Halestrap, 1987). However, it has been found that sucrose (which was used by Halestrap to change the volume of isolated mitochondria) can inhibit respiration of inner membrane vesicles apparently by a viscosity effect on membrane mobility rather than via a volume effect (Chazotte & Hackenbrock, 1988a,b).…”

“…In rat thymocytes cellular respiration rate has been reported to be insensitive to mitochondrial calcium level (Lakin-Thomas & Brand, 1988). In isolated rat brain synaptosomes, the depolarization-induced stimulation of respiration has been found to be calcium-dependent (Patel et al, 1988;Dagani et al, 1989) and calcium-independent (Kauppinen & Nicholls, 1986b;Erecinska & Dagani, 1990 Halestrap, 1989) leading to a rise in matrix [pyrophosphate] (shown in liver mitochondria exposed to micromolar calcium; Davidson & Halestrap, 1987). The raised matrix [pyro-phosphate] is suggested to stimulate K+ uptake into the mitochondria, leading to an increase in mitochondrial volume.…”

“…The raised matrix [pyro-phosphate] is suggested to stimulate K+ uptake into the mitochondria, leading to an increase in mitochondrial volume. Addition of calcium or pyrophosphate, or other agents that raise mitochondrial pyrophosphate, was found to increase the volume of isolated liver mitochondria (Davidson & Halestrap, 1987) and heart mitochondria (Halestrap, 1987). The increased mitochondrial volume stimulates the respiratory chain (by an undefined mechanism) at the level of electron flow into the ubiquinone pool, resulting in stimulation of respiration on many different substrates, particularly fatty acids, in liver and heart mitochondria (Davidson & Halestrap, 1987;Halestrap, 1987).…”

Direct thyroid hormone signalling via ADP‐ribosylation controls mitochondrial nucleotide transport and membrane leakiness by changing the conformation of the adenine nucleotide transporter

“…Also hypothyroid mitochondria show lower H + [21] and K + [38] permeability, whereas the C-conformation of the ANT has been found to promote increased permeability of the inner membrane to K + and H + [22,23]; moreover, the decreased permeability in hypothyroid preparations can be rapidly reversed by a single dose of T3 [21] which points to a" mechanism separate from alteration of membrane lipids [39]. Conversion of ANT to the C-conformation has also been shown to promote rapid loss of Ca 2+ from pre-loaded mitochondria [18] and to increase 1 ~tM Ca 2+ uptake [24]. Of relevance in this context is the observation that T3 induces calcium influx into liver cells within minutes and increases respiration and gluconeogenesis with the same kinetics leading to the proposal that, as with glucagon, the signalling involves increased mitochondrial uptake of cytosolic Ca 2+ [40].…”

“…Here we reconsider this conclusion and demonstrate that the velocity of the ANT can be reduced by more than half by nicotinamide addition to isolated mitochondria: further, addition of T3 at 10 pM restored the rate to normal and this was abolished by concomitant addition of nicotinamide. We go on to propose a model based on alteration of the relative proportions of the two distinct conformations of the ANT (see [16]) by ADP-ribosylation which may offer a molecular mechanism not only for the transport effects of T~ but also for those of oxidative stress and ADP-ribosylation inhibitors on Ca 2+ [17][18][19][20], H + [21,22], and K + [22][23][24] transfer across the mitochondrial inner membrane.…”