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CALCIUM ION ACCUMULATION AND VOLUME CHANGES OF ISOLATED LIVER MITOCHONDRIA. REVERSAL OF CALCIUM ION-INDUCED SWELLING.

ABSTRACT: 1. The excessive accumulation of Ca(2+) by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca(2+) from the mitochondria. When the Ca(2+) was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.

SUBMITTER: CROFTS AR

PROVIDER: S-EPMC1214335 | biostudies-other | 1965 May

REPOSITORIES: biostudies-other

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Project description:The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca(2+) was investigated. At 30 degrees C and pH7.0, mitochondria can maintain a steady-state pCa(2+) (0) (the negative logarithm of the free extramitochondrial Ca(2+) concentration) of 6.1 (0.8mum). This represents a true steady state, as slight displacements in pCa(2+) (0) away from 6.1 result in net Ca(2+) uptake or efflux in order to restore pCa(2+) (0) to its original value. In the absence of added permeant weak acid, the steady-state pCa(2+) (0) is virtually independent of the Ca(2+) accumulated in the matrix until 60nmol of Ca(2+)/mg of protein has been taken up. The steady-state pCa(2+) (0) is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa(2+) (0) is variable and appears to be governed by thermodynamic equilibration of Ca(2+) across a Ca(2+) uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa(2+) (0) in the region of 6 (1mum-free Ca(2+)) while accumulating Ca(2+). Permeant acids delay the build-up of the transmembrane pH gradient as Ca(2+) is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa(2+) (0) of 6. The steady-state pCa(2+) (0) is affected by temperature, incubation pH and Mg(2+). The activity of the Ca(2+) uniport, rather than that of the respiratory chain, is rate-limiting when pCa(2+) (0) is greater than 5.3 (free Ca(2+) less than 5mum). When the Ca(2+) electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa(2+) (0) (fall in free Ca(2+)). At pCa(2+) (0) 6.1, the activity of the Ca(2+) uniport is kinetically limited to 5nmol of Ca(2+)/min per mg of protein, even when the Ca(2+) electrochemical gradient is large. A steady-state cycling of Ca(2+) through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa(2+) (0) by liver mitochondria in vivo.

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CALCIUM ION ACCUMULATION AND VOLUME CHANGES OF ISOLATED LIVER MITOCHONDRIA. REVERSAL OF CALCIUM ION-INDUCED SWELLING.

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