Thyroid-hormone control of state-3 respiration in isolated rat liver mitochondria.
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ABSTRACT:
SUBMITTER: Hafner RP
PROVIDER: S-EPMC1133694 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
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Project description:1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H(+) (or OH(-)) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and beta-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5.5 and 8.5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the -->H(+)/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of -->H(+)/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the -->H(+)/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na(+). These apparent changes of -->H(+)/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial -->H(+)/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial -->H(+)/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid-base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.
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Project description:Using different conditions mitochondria from hypothyroid rats can show both unchanged ADP/O ratios and lowered ADP/O ratios without evidence of uncoupling when compared with euthyroid controls. Raising the free Ca2+ concentration to around 25 nM progressively lowered the ADP/O ratio in hypothyroid but not in euthyroid mitochondria. Ruthenium Red did not alter this behaviour and further increasing the Ca2+ concentration to levels below those which stimulate State 3 respiration had no additional effect. Measurements of the free Ca2+ concentration in the mitochondrial suspending medium using a Quin 2 fluorescence assay showed that the mitochondria did not buffer the free Ca2+ at these low concentrations. At 25 nM-free Ca2+, addition of 10-13) M-T3 to hypothyroid mitochondria produced an immediate and significant increase in the ADP/O ratio without altering the free Ca2+ concentration. The hormone effect was maximal by 10(-11) M. The concentration of ATP synthetase can be estimated to lie at about 10 nM in these experiments. Hence it appears possible that a substantial amplification of the hormone signal may have taken place. Comparison with binding studies suggests that T3 may have been maximally stimulating when somewhat less than half its receptor sites had been filled. The possible mechanisms by which this receptor mediated alteration of the ADP/O ratio might be achieved are discussed.
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